U.S. patent application number 11/894045 was filed with the patent office on 2008-09-11 for compositions and methods for intraocular delivery of fibronectin scaffold domain proteins.
This patent application is currently assigned to Adnexus, A Bristol-Myers Squibb R&D Company. Invention is credited to Yan Chen, Elena Getmanova, Jochem Gokemeijer, Alan S. Harris, Ai Ching Lim, Lin Sun, Michael Wittekind, Martin C. Wright.
Application Number | 20080220049 11/894045 |
Document ID | / |
Family ID | 39741864 |
Filed Date | 2008-09-11 |
United States Patent
Application |
20080220049 |
Kind Code |
A1 |
Chen; Yan ; et al. |
September 11, 2008 |
Compositions and methods for intraocular delivery of fibronectin
scaffold domain proteins
Abstract
The present disclosure relates to novel sustained-release
intraocular drug delivery systems and improvements in the treatment
of retinopathies. In particular, fibronectin scaffold domain
proteins that selectively inhibit VEGFR-2 are contemplated.
Inventors: |
Chen; Yan; (Lexington,
MA) ; Getmanova; Elena; (Lexington, MA) ;
Wright; Martin C.; (Boston, MA) ; Harris; Alan
S.; (Andover, MA) ; Lim; Ai Ching; (Newton,
MA) ; Gokemeijer; Jochem; (Arlington, MA) ;
Sun; Lin; (West Roxbury, MA) ; Wittekind;
Michael; (Bainbridge Island, WA) |
Correspondence
Address: |
ROPES & GRAY LLP
PATENT DOCKETING 39/41, ONE INTERNATIONAL PLACE
BOSTON
MA
02110-2624
US
|
Assignee: |
Adnexus, A Bristol-Myers Squibb
R&D Company
Waltham
MA
|
Family ID: |
39741864 |
Appl. No.: |
11/894045 |
Filed: |
August 17, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11448171 |
Jun 5, 2006 |
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11894045 |
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11101954 |
Apr 7, 2005 |
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11448171 |
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PCT/US04/40885 |
Dec 6, 2004 |
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11101954 |
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60527886 |
Dec 5, 2003 |
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Current U.S.
Class: |
424/428 ;
424/130.1; 424/133.1; 514/1.1 |
Current CPC
Class: |
A61P 9/10 20180101; A61P
27/02 20180101; C07K 16/30 20130101; C07K 2317/73 20130101; C07K
2318/20 20130101; C07K 16/2863 20130101; C07K 2317/31 20130101;
C07K 2317/92 20130101; C07K 14/00 20130101; A61K 2039/505
20130101 |
Class at
Publication: |
424/428 ;
424/133.1; 424/130.1; 514/12 |
International
Class: |
A61F 9/00 20060101
A61F009/00; A61K 39/395 20060101 A61K039/395; A61K 38/16 20060101
A61K038/16; A61P 27/02 20060101 A61P027/02; A61P 9/10 20060101
A61P009/10 |
Claims
1. A sustained-release intraocular drug delivery system comprising:
a therapeutic component comprising an antiangiogenic polypeptide
component; and a polymeric component associated with the
therapeutic component to permit the therapeutic component to be
released into the interior of an eye of an individual at a
therapeutically effective dosage for a period of time after the
drug delivery system is placed in the eye.
2. The system of claim 1 wherein said therapeutic component and
said polymeric component are combined in a form selected from the
group consisting of a) an implant device, or b) a plurality of
particles.
3. The system of claim 2 wherein the antiangiogenic polypeptide
component comprises an antibody, antibody fragment, or artificial
antibody, and humanized versions of these polypeptides.
4. The system of claim 3 wherein the antiangiogenic component
comprises an artificial antibody or a humanized version
thereof.
5. The system of claim 4 wherein the artificial antibody comprises
a scaffold region based upon a fibronectin.
6. The system of claim 5 wherein the artificial antibody comprises
fibronectin based "addressable" therapeutic binding molecule
("FATBIM").
7. The system of claim 6 wherein the FATBIM is selected from the
group consisting of CT322, C7S100 and C7C100.
8. A sustained-release intraocular drug delivery system comprising:
a therapeutic component comprising an antiangiogenic polypeptide
component, wherein the therapeutic component is selected from the
group consisting of C7S100 and C7C100; and a polymeric component
associated with the therapeutic component to permit the therapeutic
component to be released into the interior of an eye of an
individual at a therapeutically effective dosage for a period of
time after the drug delivery system is placed in the eye.
9. A method of treating a retinopathy, the method comprising
administering, to a patient in need thereof, a therapeutically
effective amount of a polypeptide that binds to human VEGFR-2, the
polypeptide comprising between about 80 and about 150 amino acids
that have a structural organization comprising: i) at least five to
seven beta strands or beta-like strands distributed among at least
two beta sheets, and ii) at least one loop portion connecting two
strands that are beta strands or beta-like strands, which loop
portion participates in binding to VEGFR-2, wherein the polypeptide
binds to an extracellular domain of the human VEGFR-2 protein with
a dissociation constant (K.sub.D) of less than 1.times.10.sup.-6 M
and inhibits VEGFR-2 mediated angiogenesis.
Description
RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S. patent
application Ser. No. 11/448,171, entitled "Inhibitors of Type 2
Vascular Endothelial Growth Factor Receptors," filed Jun. 5, 2006,
which is a continuation of International Application
PCT/US04/40885, entitled "Inhibitors of Type 2 Vascular Endothelial
Growth Factor Receptors," filed Dec. 6, 2004 and designating the
U.S., which claims the benefit of U.S. Provisional Application No.
60/527,886, entitled "Inhibitors of Vascular Endothelial Growth
Factor Receptors," filed Dec. 5, 2003. All of the teachings of the
above-referenced applications are incorporated herein by
reference.
BACKGROUND OF THE INVENTION
[0002] The present disclosure relates to novel sustained-release
intraocular drug delivery systems and methods for using these
systems to inhibit biological activities in the eye. In particular,
the systems of the invention inhibit biological activities mediated
by vascular endothelial growth factors (VEGFs).
[0003] Angiogenesis is the process by which new blood vessels are
formed from pre-existing capillaries or post capillary venules; it
is an important component of many physiological processes including
ovulation, embryonic development, wound repair, and collateral
vascular generation in the myocardium. Angiogenesis is also central
to a number of pathological conditions such as tumor growth and
metastasis, diabetic retinopathy, and macular degeneration. In many
instances, the process begins with the activation of existing
vascular endothelial cells in response to a variety of cytokines
and growth factors. In cancer, tumor released cytokines or
angiogenic factors stimulate vascular endothelial cells by
interacting with specific cell surface receptors. The activated
endothelial cells secrete enzymes that degrade the basement
membrane of the vessels, allowing invasion of the endothelial cells
into the tumor tissue. Once situated, the endothelial cells
differentiate to form new vessel offshoots of pre-existing vessels.
The new blood vessels provide nutrients to the tumor, facilitating
further growth, and also provide a route for metastasis.
[0004] To date, numerous angiogenic factors have been identified,
including the particularly potent factor VEGF. VEGF was initially
purified from the conditioned media of folliculostellate cells and
from a variety of cell lines. More recently a number of structural
homologs and alternatively spliced forms of VEGF have been
identified. The various forms of VEGF bind as high affinity ligands
to a suite of VEGF receptors (VEGFRs). VEGFRs are tyrosine kinase
receptors, many of which are important regulators of angiogenesis.
The VEGFR family includes 3 major subtypes: VEGFR-1, VEGFR-2 (also
known as Kinase Insert Domain Receptor, "KDR", in humans), and
VEGFR-3. Among VEGF forms, VEGF-A, VEGF-C and VEGF-D are known to
bind and activate VEGFR-2.
[0005] VEGF, acting through its cognate receptors, can function as
an endothelial specific mitogen during angiogenesis. In addition,
there is substantial evidence that VEGF and VEGFRs are up-regulated
in conditions characterized by inappropriate angiogenesis, such as
cancer. As a result, a great deal of research has focused on the
identification of therapeutics that target and inhibit VEGF or
VEGFR.
[0006] Vascular diseases of the eye comprise a major cause of
blindness and have only imperfect methods of treatment. These
diseases include various retinopathies and macular degeneration.
Retinopathy frequently results in blindness or severely limited
vision due to unorganized growth and/or damage to retinal blood
vessels. There are two major types of retinopathy: diabetic
retinopathy and retinopathy of prematurity. Diabetic retinopathy
affects nearly 80% of all diabetics who have had diabetes for more
than 15 years. Retinopathy of prematurity is thought to result from
oxygen toxicity, with about 15,000 premature infants a year being
diagnosed with ROP in the United States alone. Macular degeneration
results from the neovascular growth of the choroid vessel
underneath the macula. There are two types of macular degeneration:
dry and wet. While wet macular degeneration only comprises 15% of
all macular degeneration, nearly all wet macular degeneration leads
to blindness. In addition, wet macular degeneration nearly always
results from dry macular degeneration. Once one eye is affected by
wet macular degeneration, the condition almost always affects the
other eye.
[0007] Current therapeutic approaches that target or inhibit VEGF
or VEGFR include antibodies, peptides, and small molecule kinase
inhibitors. Of these, antibodies are the most widely used for in
vivo recognition and inhibition of ligands and cellular receptors.
Highly specific antibodies have been used to block receptor-ligand
interaction, thereby neutralizing the biological activity of the
components, and also to specifically deliver toxic agents to cells
expressing the cognate receptor on its surface. Although effective,
antibodies are large, complex molecules that rely on expression in
recombinant mammalian cells for production. Antibodies also cause a
variety of side effects that are often undesirable, including
activation of complement pathways and antibody-directed cellular
cytotoxicity. As a result, there remains a need for effective
therapeutics that can specifically inhibit VEGF/VEGFR pathways as a
treatment for disorders characterized by inappropriate
angiogenesis, in particular for the treatment of retinopathies.
Additionally, long-lasting treatments are in need for intraocular
treatments.
SUMMARY OF THE INVENTION
[0008] The application provides sustained-release intraocular drug
delivery systems comprising: a therapeutic component comprising an
antiangiogenic polypeptide component; and a polymeric component
associated with the therapeutic component to permit the therapeutic
component to be released into the interior of an eye of an
individual at a therapeutically effective dosage for a period of
time after the drug delivery system is placed in the eye. The
therapeutic and polymeric components may be combined in an implant
device or as a plurality of particles.
[0009] In some embodiments, the antiangiogenic polypeptide
component comprises an antibody, antibody fragment, or an
artificial antibody, such as a scaffold region based upon a
fibronectin, as well as the humanized versions thereof. An
artificial antibody may comprise fibronectin based "addressable"
therapeutic binding molecules ("FATBIM"), such as CT322, C7S100 and
C7C100. In some embodiments, the therapeutic component is selected
from the group consisting of C7S100 and C7C100; and a polymeric
component associated with the therapeutic component to permit the
therapeutic component to be released into the interior of an eye of
an individual at a therapeutically effective dosage for a period of
time after the drug delivery system is placed in the eye.
[0010] In some embodiments, the antiangiogenic polypeptide
component comprises a sequence selected from SEQ ID NOs: 6-183,
186-197, 199 and 241-310. In exemplary embodiments the sequence is
selected from SEQ ID NO: 194 or 195. In some embodiments the
polypeptide component comprises PEG.
[0011] The application further provides novel methods of treatment.
In one aspect, a method of treating a retinopathy is provided, the
method comprising administering, to a patient in need thereof, a
therapeutically effective amount of a polypeptide that binds to
human VEGFR-2, the polypeptide comprising between about 80 and
about 150 amino acids that have a structural organization
comprising: i) at least five to seven beta strands or beta-like
strands distributed among at least two beta sheets, and ii) at
least one loop portion connecting two strands that are beta strands
or beta-like strands, which loop portion participates in binding to
VEGFR-2, wherein the polypeptide binds to an extracellular domain
of the human VEGFR-2 protein with a dissociation constant (K.sub.D)
of less than 1.times.10.sup.-6 M and inhibits VEGFR-2 mediated
angiogenesis. The methods of treatment also provide for
administering to a patient in need thereof the sustained-release
intraocular drug delivery systems of the invention.
[0012] The antiangiogenic polypeptide components may comprise
single domain polypeptides. A single domain polypeptide described
herein will generally be a polypeptide that binds to a target, such
as VEGFR-2, and where target binding activity situated within a
single structural domain, as differentiated from, for example,
antibodies and single chain antibodies, where antigen binding
activity is generally contributed by both a heavy chain variable
domain and a light chain variable domain. The disclosure also
provides larger proteins that may comprise single domain
polypeptides that bind to target. For example, a plurality of
single domain polypeptides may be connected to create a composite
molecule with increased avidity. Likewise, a single domain
polypeptide may be attached (e.g., as a fusion protein) to any
number of other polypeptides. In certain aspects a single domain
polypeptide may comprise at least five to seven beta or beta-like
strands distributed among at least two beta sheets, as exemplified
by immunoglobulin and immunoglobulin-like domains. A beta-like
strand is a string of amino acids that participates in the
stabilization of a single domain polypeptide but does not
necessarily adopt a beta strand conformation. Whether a beta-like
strand participates in the stabilization of the protein may be
assessed by deleting the string or altering the sequence of the
string and analyzing whether protein stability is diminished.
Stability may be assessed by, for example, thermal denaturation and
renaturation studies. Preferably, a single domain polypeptide will
include no more than two beta-like strands. A beta-like strand will
not usually adopt an alpha-helical conformation but may adopt a
random coil structure. In the context of an immunoglobulin domain
or an immunoglobulin-like domain, a beta-like strand will most
often occur at the position in the structure that would otherwise
be occupied by the most N-terminal beta strand or the most
C-terminal beta strand. An amino acid string which, if situated in
the interior of a protein sequence would normally form a beta
strand, may, when situated at a position closer to an N- or
C-terminus, adopt a conformation that is not clearly a beta strand
and is referred to herein as a beta-like strand.
[0013] In certain embodiments, the disclosure provides single
domain polypeptides that bind to VEGFR-2. Preferably the single
domain polypeptides bind to human KDR, mouse Flk-1, or both. A
single domain polypeptide may comprise between about 80 and about
150 amino acids that have a structural organization comprising: at
least seven beta strands or beta-like strands distributed between
at least two beta sheets, and at least one loop portion connecting
two beta strands or beta-like strands, which loop portion
participates in binding to VEGFR-2. In other words a loop portion
may link two beta strands, two beta-like strands or one beta strand
and one beta-like strand. Typically, one or more of the loop
portions will participate in VEGFR-2 binding, although it is
possible that one or more of the beta or beta-like strand portions
will also participate in VEGFR-2 binding, particularly those beta
or beta-like strand portions that are situated closest to the loop
portions. A single domain polypeptide may comprise a structural
unit that is an immunoglobulin domain or an immunoglobulin-like
domain. A single domain polypeptide may bind to any part of
VEGFR-2, although polypeptides that bind to an extracellular domain
of a VEGFR-2 are preferred. Binding may be assessed in terms of
equilibrium constants (e.g., dissociation, K.sub.D) and in terms of
kinetic constants (e.g., on rate constant, k.sub.on and off rate
constant, k.sub.off). A single domain polypeptide will typically be
selected to bind to VEGFR-2 with a K.sub.D of less than 10.sup.-6M,
or less than 10.sup.-7M, 5.times.10.sup.-8M, 10.sup.-8M or less
than 10.sup.-9M. VEGFR-2 binding polypeptides may compete for
binding with one, two or more members of the VEGF family,
particularly VEGF-A, VEGF-C and VEGF-D and may inhibit one or more
VEGFR-2-mediated biological events, such as proliferation of
endothelial cells, permeabilization of blood vessels and increased
motility in endothelial cells. VEGFR-2 binding polypeptides may be
used for therapeutic purposes as well as for any purpose involving
the detection or binding of VEGFR-2. Polypeptides for therapeutic
use will generally have a K.sub.D of less than 5.times.10.sup.-8M,
less than 10.sup.-8M or less than 10.sup.-9M, although higher
K.sub.D values may be tolerated where the k.sub.off is sufficiently
low or the k.sub.on is sufficiently high. In certain embodiments, a
single domain polypeptide that binds to VEGFR-2 will comprise a
consensus VEGFR-2 binding sequence selected from the group
consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID
NO:4. Preferably, such sequence will be situated in a loop,
particularly the FG loop.
[0014] In certain embodiments, the single domain polypeptide
comprises an immunoglobulin (Ig) variable domain. The Ig variable
domain may, for example, be selected from the group consisting of:
a human V.sub.L domain, a human V.sub.H domain and a camelid
V.sub.HH domain. One, two, three or more loops of the Ig variable
domain may participate in binding to VEGFR-2, and typically any of
the loops known as CDR1, CDR2 or CDR3 will participate in VEGFR-2
binding.
[0015] In certain embodiments, the single domain polypeptide
comprises an immunoglobulin-like domain. One, two, three or more
loops of the immunoglobulin-like domain may participate in binding
to VEGFR-2. A preferred immunoglobulin-like domain is a fibronectin
type III (Fn3) domain. Such domain may comprise, in order from
N-terminus to C-terminus, a beta or beta-like strand, A; a loop,
AB; a beta strand, B; a loop, BC; a beta strand C; a loop CD; a
beta strand D; a loop DE; a beta strand F; a loop FG; and a beta or
beta-like strand G. See FIG. 22 for an example of the structural
organization. Optionally, any or all of loops AB, BC, CD, DE, EF
and FG may participate in VEGFR-2 binding, although preferred loops
are BC, DE and FG. A preferred Fn3 domain is an Fn3 domain derived
from human fibronectin, particularly the 10.sup.th Fn3 domain of
fibronectin, referred to as .sup.10Fn3. It should be noted that
none of VEGFR-2 binding polypeptides disclosed herein have an amino
acid sequence that is identical to native .sup.10Fn3; the sequence
has been modified to obtain VEGFR-2 binding proteins, but proteins
having the basic structural features of .sup.10Fn3, and
particularly those retaining recognizable sequence homology to the
native .sup.10Fn3 are nonetheless referred to herein as ".sup.10Fn3
polypeptides". This nomenclature is similar to that found in the
antibody field where, for example, a recombinant antibody V.sub.L
domain generated against a particular target protein may not be
identical to any naturally occurring V.sub.L domain but nonetheless
the protein is recognizably a V.sub.L protein. A .sup.10Fn3
polypeptide may be at least 60%, 65%, 70%, 75%, 80%, 85%, or 90%
identical to the human .sup.10Fn3 domain, shown in SEQ ID NO:5.
Much of the variability will generally occur in one or more of the
loops. Each of the beta or beta-like strands of a .sup.10Fn3
polypeptide may consist essentially of an amino acid sequence that
is at least 80%, 85%, 90%, 95% or 100% identical to the sequence of
a corresponding beta or beta-like strand of SEQ ID NO: 5, provided
that such variation does not disrupt the stability of the
polypeptide in physiological conditions. A .sup.10Fn3 polypeptide
may have a sequence in each of the loops AB, CD, and EF that
consists essentially of an amino acid sequence that is at least
80%, 85%, 90%, 95% or 100% identical to the sequence of a
corresponding loop of SEQ ID NO:5. In many instances, any or all of
loops BC, DE, and FG will be poorly conserved relative to SEQ ID
NO:5. For example, all of loops BC, DE, and FG may be less than
20%, 10%, or 0% identical to their corresponding loops in SEQ ID
NO:5.
[0016] In certain embodiments, the disclosure provides a
non-antibody polypeptide comprising a domain having an
immunoglobulin-like fold that binds to VEGFR-2. The non-antibody
polypeptide may have a molecular weight of less than 20 kDa, or
less than 15 kDa and will generally be derived (by, for example,
alteration of the amino acid sequence) from a reference, or
"scaffold", protein, such as an Fn3 scaffold. The non-antibody
polypeptide may bind VEGFR-2 with a K.sub.D less than 10.sup.-6M,
or less than 10.sup.-7M, less than 5.times.10.sup.-8M, less than
10.sup.-8M or less than 10.sup.-9M. The unaltered reference protein
either will not meaningfully bind to VEGFR-2 or will bind with a
K.sub.D of greater than 10.sup.-6M. The non-antibody polypeptide
may inhibit VEGF signaling, particularly where the non-antibody
polypeptide has a K.sub.D of less than 5.times.10.sup.-8M, less
than 10.sup.-8M or less than 10.sup.-9M, although higher K.sub.D
values may be tolerated where the k.sub.off is sufficiently low
(e.g., less than 5.times.10.sup.-4 s.sup.-1). The
immunoglobulin-like fold may be a .sup.10Fn3 polypeptide.
[0017] In certain embodiments, the disclosure provides a
polypeptide comprising a single domain having an immunoglobulin
fold that binds to VEGFR-2. The polypeptide may have a molecular
weight of less than 20 kDa, or less than 15 kDa and will generally
be derived (by, for example, alteration of the amino acid sequence)
from a variable domain of an immunoglobulin. The polypeptide may
bind VEGFR-2 with a K.sub.D less than 10.sup.-6M, or less than
10.sup.-7M, less than 5.times.10.sup.-8M, less than 10.sup.-8M or
less than 10.sup.-9M. The polypeptide may inhibit VEGF signaling,
particularly where the polypeptide has a K.sub.D of less than
5.times.10.sup.-8M, less than 10.sup.-8M or less than 10.sup.-9M,
although higher K.sub.D values may be tolerated where the k.sub.off
is sufficiently low or where the k.sub.on is sufficiently high. In
certain preferred embodiments, a single domain polypeptide having
an immunoglobulin fold derived from an immunoglobulin light chain
variable domain and capable of binding to VEGFR-2 may comprise an
amino acid sequence selected from the group consisting of: SEQ ID
NOs:241-310.
[0018] In certain preferred embodiments, the disclosure provides
VEGFR-2 binding polypeptides comprising the amino acid sequence of
any of SEQ ID NOs:192-194. In the case of a polypeptide comprising
the amino acid sequence of SEQ ID NO:194, a PEG moiety or other
moiety of interest, may be covalently bound to the cysteine at
position 93. The PEG moiety may also be covalently bonded to an
amine moiety in the polypeptide. The amine moiety may be, for
example, a primary amine found at the N-terminus of a polypeptide
or an amine group present in an amino acid, such as lysine or
arginine. In certain embodiments, the PEG moiety is attached at a
position on the polypeptide selected from the group consisting of:
a) the N-terminus; b) between the N-terminus and the most
N-terminal beta strand or beta-like strand; c) a loop positioned on
a face of the polypeptide opposite the target-binding site; d)
between the C-terminus and the most C-terminal beta strand or
beta-like strand; and e) at the C-terminus.
[0019] In certain aspects, the disclosure provides short peptide
sequences that mediate VEGFR-2 binding. Such sequences may mediate
VEGFR-2 binding in an isolated form or when inserted into a
particular protein structure, such as an immunoglobulin or
immunoglobulin-like domain. Examples of such sequences include
those disclosed as SEQ ID NOs:1-4 and other sequences that are at
least 85%, 90%, or 95% identical to SEQ ID NOs:1-4 and retain
VEGFR-2 binding activity. Accordingly, the disclosure provides
substantially pure polypeptides comprising an amino acid sequence
that is at least 85% identical to the sequence of any of SEQ ID
NOs:1-4, wherein said polypeptide binds to a VEGFR-2 and competes
with a VEGF species for binding to VEGFR-2. Examples of such
polypeptides include a polypeptide comprising an amino acid
sequence that is at least 80%, 85%, 90%, 95% or 100% identical to
an amino acid sequence at least 85% identical to the sequence of
any of SEQ ID NOs:6-183, 186-197, 199 and 311-528. Preferably such
polypeptide will inhibit a biological activity of VEGF and may bind
to VEGFR-2 with a K.sub.D less than 10.sup.-6M, or less than
10.sup.-7M, less than 5.times.10.sup.-8M, less than 10.sup.-8M or
less than 10.sup.-9M.
[0020] In certain embodiments, any of the VEGFR-2 binding
polypeptides described herein may be bound to one or more
additional moieties, including, for example, a moiety that also
binds to VEGFR-2 (e.g., a second identical or different VEGFR-2
binding polypeptide), a moiety that binds to a different target
(e.g., to create a dual-specificity binding agent), a labeling
moiety, a moiety that facilitates protein purification or a moiety
that provides improved pharmacokinetics. Improved pharmacokinetics
may be assessed according to the perceived therapeutic need. Often
it is desirable to increase bioavailability and/or increase the
time between doses, possibly by increasing the time that a protein
remains available in the serum after dosing. In some instances, it
is desirable to improve the continuity of the serum concentration
of the protein over time (e.g., decrease the difference in serum
concentration of the protein shortly after administration and
shortly before the next administration). Moieties that tend to slow
clearance of a protein from the blood include polyethylene glycol,
sugars (e.g. sialic acid), and well-tolerated protein moieties
(e.g., Fc fragment or serum albumin). The single domain polypeptide
may be attached to a moiety that reduces the clearance rate of the
polypeptide in a mammal (e.g., mouse, rat, or human) by greater
than three-fold relative to the unmodified polypeptide. Other
measures of improved pharmacokinetics may include serum half-life,
which is often divided into an alpha phase and a beta phase. Either
or both phases may be improved significantly by addition of an
appropriate moiety. Where polyethylene glycol is employed, one or
more PEG molecules may be attached at different positions in the
protein, and such attachment may be achieved by reaction with
amines, thiols or other suitable reactive groups. Pegylation may be
achieved by site-directed pegylation, wherein a suitable reactive
group is introduced into the protein to create a site where
pegylation preferentially occurs. In a preferred embodiment, the
protein is modified so as to have a cysteine residue at a desired
position, permitting site directed pegylation on the cysteine. PEG
may vary widely in molecular weight and may be branched or linear.
Notably, the present disclosure establishes that pegylation is
compatible with target binding activity of .sup.10Fn3 polypeptides
and, further, that pegylation does improve the pharmacokinetics of
such polypeptides. Accordingly, in one embodiment, the disclosure
provides pegylated forms of .sup.10Fn3 polypeptides, regardless of
the target that can be bound by such polypeptides.
[0021] In certain embodiments, the disclosure provides a
formulation comprising any of the VEGFR-2 binding polypeptides
disclosed herein. A formulation may be a therapeutic formulation
comprising a VEGFR-2 binding polypeptide and a pharmaceutically
acceptable carrier. A formulation may also be a combination
formulation, comprising an additional active agent, such as an
anti-cancer agent or an anti-angiogenic agent.
[0022] In certain aspects, the disclosure provides methods for
using a VEGFR-2 binding protein to inhibit a VEGF biological
activity in a cell or to inhibit a biological activity mediated by
VEGFR-2. The cell may be situated in vivo or ex vivo, and may be,
for example, a cell of a living organism, a cultured cell or a cell
in a tissue sample. The method may comprise contacting said cell
with any of the VEGFR-2-inhibiting polypeptides disclosed herein,
in an amount and for a time sufficient to inhibit such biological
activity.
[0023] In certain aspects, the disclosure provides methods for
treating a subject having a condition which responds to the
inhibition of VEGF or VEGFR-2. Such a method may comprise
administering to said subject an effective amount of any of the
VEGFR-2 inhibiting polypeptides described herein. A condition may
be one that is characterized by inappropriate angiogenesis. A
condition may be a hyperproliferative condition. Examples of
conditions (or disorders) suitable for treatment include autoimmune
disorders, inflammatory disorders, retinopathies (particularly
proliferative retinopathies), and cancers. Any of the VEGFR-2
inhibiting polypeptides described herein may be used for the
preparation of a medicament for the treatment of a disorder,
particularly a disorder selected from the group consisting of: an
autoimmune disorder, an inflammatory disorder, a retinopathy, and a
cancer.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] FIGS. 1A-1D are graphs and images depicting the
characterization of KDR-binding single clones from Round 6 of KDR
selection. FIG. 1A is a graph showing the specific binding of
fibronectin-based binding proteins to 25 nM of KDR-Fc analyzed in
radioactive equilibrium binding assay. FIG. 1B is a graph showing
the inhibition of specific binding of KDR-Fc and selected
fibronectin based binding proteins in the presence of 100-fold
excess of VEGF.sub.165. As shown in this figure, certain binding
proteins bound KDR-Fc competitively with VEGF.sub.165 while others,
exemplified by clone 8, did not compete with VEGF.sub.165. FIG. 1C
is a graph showing the inhibition of KDR-Fc interaction with
immobilized VEGF.sub.165 in presence of selected fibronectin based
binding proteins analyzed in BIAcore. FIG. 1D is an image showing
binding of VR28 to KDR-expressing and control cells detected by
immunofluorescence.
[0025] FIG. 2 is a graph showing the selection profile for the
affinity maturation of VR28 KDR binder. Shown at left is binding of
the VR28 clone to KDR-Fc and Flk1-Fc (very low, unlabeled bar).
Shown at center is binding of a crude mutagenized pool and
subsequent enrichment rounds to KDR-Fc. Shown at right is binding
of further enrichment rounds to Flk-1-Fc. Binding was estimated in
radioactive equilibrium binding assay as a percentage of input,
using 1 nM KDR-Fc or Flk1-Fc.
[0026] FIGS. 3A and 3B are graphs depicting the characterization of
KDR-binding single clones from Round 4 of anti-KDR affinity
maturation of VR28 binder. FIG. 3A shows the saturation binding of
VR28 (-.box-solid.-) and affinity matured K1 (-.tangle-solidup.-),
K6 (--), K9 (-.diamond-solid.-), K10 (- -), K12 K13 (-.DELTA.-),
K14 (-.gradient.-), K15 (-.diamond.-) to KDR-Fc in radioactive
equilibrium binding assay FIG. 3B shows the binding of clones with
and without N-terminal deletion to KDR-Fc. Deletion .DELTA.1-8 in
the N-terminus of fibronectin-based binding proteins improved
binding to KDR-Fc. The data represents an average KDR-Fc binding of
23 independent clones with and without N-terminal deletion.
[0027] FIG. 4 is a graph showing the binding of the selected clones
to KDR and Flk-1. Specific binding of VR28 and selected clones
after four rounds of affinity maturation to human KDR (K clones)
and seven rounds of affinity maturation to human (KDR) and mouse
(flk-1) (E clones). VEGFR-2-Fc chimeras were compared in
radioactive equilibrium binding assay. The data represents an
average of 3 independent experiments. As shown here, maturation
against both mouse and human VEGFR-2 proteins produces binders that
bind to both proteins.
[0028] FIGS. 5A and 5B are graphs showing the characterization of
VEGFR-2-binding single clones from Round 7 of affinity maturation
of VR28 binder. Saturation binding of VR28 (-.box-solid.-) and
specificity matured E3 (-.tangle-solidup.-), E5 (--), E6
(-.diamond-solid.-), E9 (- -), E18 E19 (-.DELTA.-), E25
(-.gradient.-), E26 (-.diamond.-), E28 (-.largecircle.-), E29 (-X-)
clones to KDR (FIG. 5A) and Flk1 (FIG. 5B)-Fc chimeras was tested
in radioactive equilibrium binding assay.
[0029] FIGS. 6A and 6B are graphs showing the characterization of
VEGFR-2 binding by single clones from Round 7 of affinity
maturation of the VR28 binder. FIG. 6A shows the importance of
arginine at positions 79 and 82 in binders with dual specificity to
human and mouse VEGFR-2 for binding to mouse VEGFR-2 (Flk1). When
either of these positions was replaced by amino acid other than R
(X79=E, Q, W, P; X82=L, K), binding to Flk1 but not to KDR
significantly decreased. FIG. 6B shows the importance of all three
variable loops (BC, DE and FG) of KDR fibronectin-based binding
proteins for binding to the target in these proteins. Substitution
of each loop at a time by NNS sequence affected binding to KDR and
Flk1. The binding data is an average from E6 and E26 clones.
[0030] FIGS. 7A and 7B are graphs showing the binding of selected
fibronectin-based binding proteins to CHO cells expressing human
KDR receptor (FIG. 7A) and EpoR-Flk1 chimera (FIG. 7B). E18
(-.box-solid.-), E19 (-.tangle-solidup.-), E26 (--), E29
(-.diamond-solid.-) and WT fibronectin-based scaffold proteins were
tested. No binding to control CHO cells was observed (data not
shown).
[0031] FIGS. 8A and 8B are graphs showing the inhibition of
VEGF-induced proliferation of Ba/F3-KDR (FIG. 8A) and Ba/F3-Flk1
(FIG. 8B) cells, expressing KDR and Flk1 in the presence of
different amounts of fibronectin-based binding proteins: E18
(-.box-solid.-), E19 (-.tangle-solidup.-), E26
(-.tangle-solidup.-), E29 (-.diamond-solid.-), M5 (- -), WT and
anti-KDR or anti-flk-1 Ab (-.DELTA.-). The data represents an
average of 2 independent experiments.
[0032] FIG. 9 is a graph showing the results of a HUVEC
proliferation assay in the presence of different amounts of
fibronectin-based scaffold proteins: E18 (-.box-solid.-), E19
(-.tangle-solidup.-), E26 (--), E29 (-.diamond-solid.-), M5 (- -),
WT The data represents an average of 2 independent experiments. As
shown, the KDR binding proteins caused a decrease in proliferation
by approximately 40%.
[0033] FIG. 10 is a set of graphs showing the reversible refolding
of M5FL in optimized buffer.
[0034] FIG. 11 is an image showing SDS-PAGE analysis of pegylated
forms of M5FL. M, molecular weight markers [Sea Blue Plus,
Invitrogen]; -, M5FL alone; 20, M5FL with 20 kD PEG; 40, M5FL with
40 kD PEG.
[0035] FIG. 12 is a graph showing the inhibition of VEGF-induced
proliferation of Ba/F3-KDR cells with differing amounts of M5FL
(-.diamond-solid.-), M5FL PEG20 (-.box-solid.-) and M5FL
PEG40(-.tangle-solidup.-), respectively. Pegylation has little or
no effect on M5FL activity in this assay.
[0036] FIG. 13 shows western analysis of VEGFR-2 signaling in
endothelial cells. Phospho VEGFR-2--Visualization of phosphorylated
VEGFR-2. VEGFR-2--Sample loading control. Phospho
ERK1/2--Visualization of phosphorylated ERK1/2 (MAPK). ERK1-Sample
loading control. The results demonstrated that 130 pM CT-01 blocks
VEGFR-2 activation and signaling by VEGF-A.
[0037] FIG. 14 shows that various .sup.10Fn3-derived molecules
(e.g. M5FL, F10, CT-01) can block VEGF-A and VEGF-D signaling.
[0038] FIG. 15 shows a comparison of .sup.125I native, pegylated
CT-01 administered i.v. & i.p. CT-01 is a 12 kDa protein. It is
rapidly cleared from the blood. Addition of a 40 kDa PEG reduces
its clearance rate and increases the AUC by 10 fold. Half life of
16 hr in rats is equivalent to 2.times. dosing per week in humans.
Administration route: i.p. CT-01-PEG40 has an AUC that is only 50%
of an i.v. administration.
[0039] FIG. 16 shows the tissue distribution of .sup.125I-CT01PEG40
in normal rats. Tissue distribution of .sup.125I-CT01PEG40
indicates secretion primarily via the liver and secondarily via the
kidney. This is expected for the high molecular weight PEG form. No
long term accumulation of CT-01PEG40 is detected.
[0040] FIG. 17 is a schematic view of the Miles Assay that measures
vascular permeability.
[0041] FIG. 18 shows that CT-01 blocks VEGF in vivo using the Miles
Assay. The results indicate that 5 mg/kg of CT01-PEG40 blocks VEGF
challenge.
[0042] FIG. 19 shows that CT-01 inhibits tumor growth using the
B16-F10 Murine Melanoma Tumor Assay.
[0043] FIG. 20 shows that CT-01 inhibits tumor growth using U87
Human Glioblastoma.
[0044] FIGS. 21A and 21B show the sequences of VEGFR binding
polypeptides that are based on an antibody light chain
framework/scaffold (SEQ ID NOs:241-310).
[0045] FIG. 22 shows the structural organization for a single
domain polypeptide having an immunoglobulin fold (a V.sub.H domain
of an immunoglobulin, left side) and a single domain polypeptide
having an immunoglobulin-like fold (a .sup.10Fn3 domain, right
side).
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0046] A "functional Fc region" possesses at least one "effector
function" of a native sequence Fc region. Exemplary "effector
functions" include C1q binding; complement dependent cytotoxicity
(CDC); Fc receptor binding; antibody-dependent cell-mediated
cytotoxicity (ADCC); phagocytosis; down regulation of cell surface
receptors (e.g. B cell receptor; BCR), etc. Such effector functions
generally require the Fc region to be combined with a binding
domain (e.g. an antibody variable domain) and can be assessed using
various assays known in the art for evaluating such antibody
effector functions.
[0047] A "native sequence Fc region" comprises an amino acid
sequence identical to the amino acid sequence of an Fc region found
in nature.
[0048] A "variant Fc region" comprises an amino acid sequence which
differs from that of a native sequence Fc region by virtue of at
least one amino acid modification. Preferably, the variant Fc
region has at least one amino acid substitution compared to a
native sequence Fc region or to the Fc region of a parent
polypeptide, e.g. from about one to about ten amino acid
substitutions, and preferably from about one to about five amino
acid substitutions in a native sequence Fc region or in the Fc
region of the parent polypeptide. The variant Fc region herein will
preferably possess at least about 80% sequence identity with a
native sequence Fc region and/or with an Fc region of a parent
polypeptide, and most preferably at least about 90% sequence
identity therewith, more preferably at least about 95% sequence
identity therewith.
[0049] "Antibody-dependent cell-mediated cytotoxicity" and "ADCC"
refer to a cell-mediated reaction in which nonspecific cytotoxic
cells that express Fc receptors (FcRs) (e.g. Natural Killer (NK)
cells, neutrophils, and macrophages) recognize bound antibody on a
target cell and subsequently cause lysis of the target cell. The
primary cells for mediating ADCC, NK cells, express Fc.gamma.RIII
only, whereas monocytes express Fc.gamma.RI, Fc.gamma.RII and
Fc.gamma.RIII. FcR expression on hematopoietic cells is summarized
in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol
9:457-92 (1991). To assess ADCC activity of a molecule of interest,
an in vitro ADCC assay, such as that described in U.S. Pat. No.
5,500,362 or 5,821,337 may be performed. Useful effector cells for
such assays include peripheral blood mononuclear cells (PBMC) and
Natural Killer (NK) cells. Alternatively, or additionally, ADCC
activity of the molecule of interest may be assessed in vivo, e.g.,
in a animal model such as that disclosed in Clynes et al. PNAS
(USA) 95:652-656 (1998).
[0050] "Human effector cells" are leukocytes which express one or
more FcRs and perform effector functions. Preferably, the cells
express at least Fc.gamma.RIII and perform ADCC effector function.
Examples of human leukocytes which mediate ADCC include peripheral
blood mononuclear cells (PBMC), natural killer (NK) cells,
monocytes, cytotoxic T cells and neutrophils; with PBMCs and NK
cells being preferred. The effector cells may be isolated from a
native source thereof, e.g. from blood or PBMCs as described
herein.
[0051] The terms "Fc receptor" and "FcR" are used to describe a
receptor that binds to the Fc region of an antibody. The preferred
FcR is a native sequence human FcR. Moreover, a preferred FcR is
one which binds an IgG antibody (a gamma receptor) and includes
receptors of the Fc.gamma.RI, Fc.gamma.RII, and Fc.gamma.RIII
subclasses, including allelic variants and alternatively spliced
forms of these receptors. Fc.gamma.RII receptors include
Fc.gamma.RIIA (an "activating receptor") and Fc.gamma.RIIB (an
"inhibiting receptor"), which have similar amino acid sequences
that differ primarily in the cytoplasmic domains thereof.
Activating receptor Fc.gamma.RIIA contains an immunoreceptor
tyrosine-based activation motif (ITAM) in its cytoplasmic domain.
Inhibiting receptor Fc.gamma.RIIB contains an immunoreceptor
tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain
(reviewed in Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs
are reviewed in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92
(1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et
al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including
those to be identified in the future, are encompassed by the term
"FcR" herein. The term also includes the neonatal receptor, FcRn,
which is responsible for the transfer of maternal IgGs to the fetus
(Guyer et al., J. Immunol. 117:587 (1976); and Kim et al., J.
Immunol. 24:249 (1994)).
[0052] "Percent (%) amino acid sequence identity" herein is defined
as the percentage of amino acid residues in a candidate sequence
that are identical with the amino acid residues in a selected
sequence, after aligning the sequences and introducing gaps, if
necessary, to achieve the maximum percent sequence identity, and
not considering any conservative substitutions as part of the
sequence identity. Alignment for purposes of determining percent
amino acid sequence identity can be achieved in various ways that
are within the skill in the art, for instance, using publicly
available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2
or Megalign (DNASTAR) software. Those skilled in the art can
determine appropriate parameters for measuring alignment, including
any algorithms needed to achieve maximal alignment over the
full-length of the sequences being compared. For purposes herein,
however, % amino acid sequence identity values are obtained as
described below by using the sequence comparison computer program
ALIGN-2. The ALIGN-2 sequence comparison computer program was
authored by Genentech, Inc. has been filed with user documentation
in the U.S. Copyright Office, Washington D.C., 20559, where it is
registered under U.S. Copyright Registration No. TXU510087, and is
publicly available through Genentech, Inc., South San Francisco,
Calif. The ALIGN-2 program should be compiled for use on a UNIX
operating system, preferably digital UNIX V4.0D. All sequence
comparison parameters are set by the ALIGN-2 program and do not
vary.
[0053] For purposes herein, the % amino acid sequence identity of a
given amino acid sequence A to, with, or against a given amino acid
sequence B (which can alternatively be phrased as a given amino
acid sequence A that has or comprises a certain % amino acid
sequence identity to, with, or against a given amino acid sequence
B) is calculated as follows: 100 times the fraction X/Y where X is
the number of amino acid residues scored as identical matches by
the sequence alignment program ALIGN-2 in that program's alignment
of A and B, and where Y is the total number of amino acid residues
in B. It will be appreciated that where the length of amino acid
sequence A is not equal to the length of amino acid sequence B, the
% amino acid sequence identity of A to B will not equal the % amino
acid sequence identity of B to A.
[0054] A "polypeptide chain" is a polypeptide wherein each of the
domains thereof is joined to other domain(s) by peptide bond(s), as
opposed to non-covalent interactions or disulfide bonds.
[0055] An "isolated" polypeptide is one that has been identified
and separated and/or recovered from a component of its natural
environment. Contaminant components of its natural environment are
materials that would interfere with diagnostic or therapeutic uses
for the polypeptide, and may include enzymes, hormones, and other
proteinaceous or nonproteinaceous solutes. In preferred
embodiments, the polypeptide will be purified (1) to greater than
95% by weight of polypeptide as determined by the Lowry method, and
most preferably more than 99% by weight, (2) to a degree sufficient
to obtain at least 15 residues of N-terminal or internal amino acid
sequence by use of a spinning cup sequenator, or (3) to homogeneity
by SDS-PAGE under reducing or nonreducing conditions using
Coomassie blue or, preferably, silver stain. Isolated polypeptide
includes the polypeptide in situ within recombinant cells since at
least one component of the polypeptide's natural environment will
not be present. Ordinarily, however, isolated polypeptide will be
prepared by at least one purification step.
[0056] Targets may also be fragments of said targets. Thus a target
is also a fragment of said target, capable of eliciting an immune
response. A target is also a fragment of said target, capable of
binding to a single domain antibody raised against the full length
target.
[0057] A fragment as used herein refers to less than 100% of the
sequence (e.g., 99%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%
etc.), but comprising 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25 or more amino acids. A fragment is
of sufficient length such that the interaction of interest is
maintained with affinity of 1.times.10.sup.-6M or better.
[0058] A fragment as used herein also refers to optional
insertions, deletions and substitutions of one or more amino acids
which do not substantially alter the ability of the target to bind
to a single domain antibody raised against the wild-type target.
The number of amino acid insertions deletions or substitutions is
preferably up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,
33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,
50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,
67, 68, 69 or 70 amino acids.
[0059] A protein of the invention that "induces cell death" is one
which causes a viable cell to become nonviable. The cell is
generally one which expresses the antigen to that the protein
binds, especially where the cell overexpresses the antigen.
Preferably, the cell is a cancer cell, e.g. a breast, ovarian,
stomach, endometrial, salivary gland, lung, kidney, colon, thyroid,
pancreatic or bladder cell. In vitro, the cell may be a SKBR3,
BT474, Calu 3, MDA-MB453, MDA-MB-361 or SKOV3 cell. Cell death in
vitro may be determined in the absence of complement and immune
effector cells to distinguish cell death induced by antibody
dependent cell-mediated cytotoxicity (ADCC) or complement dependent
cytotoxicity (CDC). Thus, the assay for cell death may be performed
using heat inactivated serum (i.e. in the absence of complement)
and in the absence of immune effector cells. To determine whether
the protein of the invention is able to induce cell death, loss of
membrane integrity as evaluated by uptake of propidium iodide (PI),
trypan blue (see Moore et al. Cytotechnology 17:1-11 (1995)) or
7AAD can be assessed relative to untreated cells.
[0060] A protein of the invention that "induces apoptosis" is one
that induces programmed cell death as determined by binding of
apoptosis related molecules or events, such as annexin V,
fragmentation of DNA, cell shrinkage, dilation of endoplasmic
reticulum, cell fragmentation, and/or formation of membrane
vesicles (called apoptotic bodies). The cell is one which expresses
the antigen to which the protein binds and may be one which
overexpresses the antigen. The cell may be a tumor cell, e.g. a
breast, ovarian, stomach, endometrial, salivary gland, lung,
kidney, colon, thyroid, pancreatic or bladder cell. In vitro, the
cell may be a SKBR3, BT474, Calu 3 cell, MDA-MB453, MDA-MB-361 or
SKOV3 cell. Various methods are available for evaluating the
cellular events associated with apoptosis. For example,
phosphatidyl serine (PS) translocation can be measured by annexin
binding; DNA fragmentation can be evaluated through DNA laddering
as disclosed in the example herein; and nuclear/chromatin
condensation along with DNA fragmentation can be evaluated by any
increase in hypodiploid cells. Preferably, the protein that induces
apoptosis is one which results in about 2 to 50 fold, preferably
about 5 to 50 fold, and most preferably about 10 to 50 fold,
induction of annexin binding relative to untreated cell in an
annexin binding assay using cells expressing the antigen to which
the protein of the invention binds.
[0061] The term "therapeutically effective amount" refers to an
amount of a drug effective to treat a disease or disorder in a
mammal. In the case of cancer, the therapeutically effective amount
of the drug may reduce the number of cancer cells; reduce the tumor
size; inhibit (i.e., slow to some extent and preferably stop)
cancer cell infiltration into peripheral organs; inhibit (i.e.,
slow to some extent and preferably stop) tumor metastasis; inhibit,
to some extent, tumor growth; and/or relieve to some extent one or
more of the symptoms associated with the disorder. To the extent
the drug may prevent growth and/or kill existing cancer cells, it
may be cytostatic and/or cytotoxic. For cancer therapy, efficacy in
vivo can, for example, be measured by assessing the time to disease
progression (TTP) and/or determining the response rates (RR).
[0062] The term "PK" is an acronym for "pharmokinetic" and
encompasses properties of a compound including, by way of example,
absorbtion, distribution, metabolism, and elimination by a subject.
A "PK modulation protein" refers to any protein or peptide that
affects the pharmokinetic properties of a biologically active
molecule when fused to or administered together with the
biologically active molecule. Examples of a PK modulation protein
include PEG, as well as human serum albumin (HSA) binders as
disclosed in US Pat. App. Nos. 20050287153 and 20070003549.
[0063] As used herein, "ocular" refers to the eye, surrounding
tissues, and to bodily fluids in the region of the eye.
Specifically, the term includes the cornea, the sclera, the uvea,
the conjunctiva (e.g., bulbar conjunctiva, palpebral conjunctiva,
and tarsal conjunctiva), anterior chamber, lacrimal sac, lacrimal
canals, lacrimal ducts, medial canthus, nasolacrimal duct, and the
eyelids (e.g., upper eyelid and lower eyelid). Additionally, the
term includes the inner surface of the eye (conjunctiva overlying
the sclera), and the inner surface of the eyelids (palpepral
conjunctiva).
Overview
[0064] The present application provides novel sustained-release
intraocular drug delivery systems that are particularly useful in
treating disorders of the eye. In exemplary embodiments, the drug
delivery systems deliver VEGFR-2 specific inhibitors
intraocularly.
[0065] In one aspect, sustained-release intraocular drug delivery
system is provided comprising a therapeutic component and a
polymeric component. The therapeutic component comprises an
antiangiogenic polypeptide component such as an antibody, an
antibody fragment, or an artificial antibody, as well as humanized
versions.
Polymeric Component
[0066] In some embodiments, the polymeric component of the
sustained-release drug delivery system comprises monomers such as
organic esters or ethers, which when degraded result in
physiologically acceptable degradation products. Anhydrides,
amides, orthoesters, or the like, by themselves or in combination
with other monomers, may also be used. The polymers are generally
condensation polymers. The polymers can be crosslinked or
non-crosslinked. If crosslinked, they are usually not more than
lightly crosslinked, and are less than 5% crosslinked, usually less
than 1% crosslinked.
[0067] In addition to carbon and hydrogen, the polymers will
include oxygen and nitrogen, particularly oxygen. The oxygen may be
present as oxy, e.g., hydroxy or ether, carbonyl, e.g.,
non-oxo-carbonyl, such as carboxylic acid ester, and the like. The
nitrogen can be present as amide, cyano, and amino. An exemplary
list of biodegradable polymers that can be used are described in
Heller, Biodegradable Polymers in Controlled Drug Delivery, In:
"CRC Critical Reviews in Therapeutic Drug Carrier Systems," Vol. 1.
CRC Press, Boca Raton, Fla. (1987).
[0068] Of particular interest are polymers of hydroxyaliphatic
carboxylic acids, either homo- or copolymers, and polysaccharides.
Included among the polyesters of interest are homo- or copolymers
of D-lactic acid, L-lactic acid, racemic lactic acid, glycolic
acid, caprolactone, and combinations thereof. Copolymers of
glycolic and lactic acid are of particular interest, where the rate
of biodegradation is controlled by the ratio of glycolic to lactic
acid. The percent of each monomer in poly(lactic-co-glycolic)acid
(PLGA) copolymer may be 0-100%, about 15-85%, about 25-75%, or
about 35-65%. In certain variations, 25/75 PLGA and/or 50/50 PLGA
copolymers are used. In other variations, PLGA copolymers are used
in conjunction with polylactide polymers.
[0069] Biodegradable polymer matrices that include mixtures of
hydrophilic and hydrophobic ended PLGA may also be employed, and
are useful in modulating polymer matrix degradation rates.
Hydrophobic ended (also referred to as capped or end-capped) PLGA
has an ester linkage hydrophobic in nature at the polymer terminus.
Typical hydrophobic end groups include, but are not limited to
alkyl esters and aromatic esters. Hydrophilic ended (also referred
to as uncapped) PLGA has an end group hydrophilic in nature at the
polymer terminus. PLGA with a hydrophilic end groups at the polymer
terminus degrades faster than hydrophobic ended PLGA because it
takes up water and undergoes hydrolysis at a faster rate (Tracy et
al., Biomaterials 20: 1057-1062 (1999)). Examples of suitable
hydrophilic end groups that may be incorporated to enhance
hydrolysis include, but are not limited to, carboxyl, hydroxyl, and
polyethylene glycol. The specific end group will typically result
from the initiator employed in the polymerization process. For
example, if the initiator is water or carboxylic acid, the
resulting end groups will be carboxyl and hydroxyl. Similarly, if
the initiator is a monofunctional alcohol, the resulting end groups
will be ester or hydroxyl.
[0070] Further polymers and polymer blends useful in the invention
can be found in U.S. Patent Applications 20060210604, 20070088014,
and 20070059336 hereby incorporated by reference.
Antiangiogenic Polypeptides
[0071] In some embodiments, the antiangiogenic polypeptide
component comprises one or more single domain polypeptides that may
be derived from two related groups of protein structures: those
proteins having an immunoglobulin fold, such as an antibody, and
those proteins having an immunoglobulin-like fold, such as an
artificial antibody. "Artificial antibody" is meant to include
fibronectin based scaffold proteins such as the Adnectins.TM. or
the "addressable" therapeutic binding molecules. By a "polypeptide"
is meant any sequence of two or more amino acids, regardless of
length, post-translation modification, or function. "Polypeptide,"
"peptide," and "protein" are used interchangeably herein.
Polypeptides can include natural amino acids and non-natural amino
acids such as those described in U.S. Pat. No. 6,559,126,
incorporated herein by reference. Polypeptides can also be modified
in any of a variety of standard chemical ways (e.g., an amino acid
can be modified with a protecting group; the carboxy-terminal amino
acid can be made into a terminal amide group; the amino-terminal
residue can be modified with groups to, e.g., enhance
lipophilicity; or the polypeptide can be chemically glycosylated or
otherwise modified to increase stability or in vivo half-life).
Polypeptide modifications can include the attachment of another
structure such as a cyclic compound or other molecule to the
polypeptide and can also include polypeptides that contain one or
more amino acids in an altered configuration (i.e., R or S; or, L
or D). The term "single domain polypeptide" is used to indicate
that the target binding activity (e.g., VEGFR-2 binding activity)
of the subject polypeptide is situated within a single structural
domain, as differentiated from, for example, antibodies and single
chain antibodies, where antigen binding activity is generally
contributed by both a heavy chain variable domain and a light chain
variable domain. It is contemplated that a plurality of single
domain polypeptides of the sort disclosed herein could be connected
to create a composite molecule with increased avidity. Likewise, a
single domain polypeptide may be attached (e.g., as a fusion
protein) to any number of other polypeptides, such as fluorescent
polypeptides, targeting polypeptides and polypeptides having a
distinct therapeutic effect.
[0072] Single domain polypeptides of either the immunoglobulin or
immunoglobulin-like scaffold will tend to share certain structural
features. For example, the polypeptide may comprise between about
80 and about 150 amino acids, which amino acids are structurally
organized into a set of beta or beta-like strands, forming beta
sheets, where the beta or beta-like strands are connected by
intervening loop portions. The beta sheets form the stable core of
the single domain polypeptides, while creating two "faces" composed
of the loops that connect the beta or beta-like strands. As
described herein, these loops can be varied to create customized
ligand binding sites, and, with proper control, such variations can
be generated without disrupting the overall stability of the
protein. In antibodies, three of these loops are the well-known
Complementarity Determining Regions (or "CDRs").
[0073] Scaffolds for formation of a single domain polypeptides
should be highly soluble and stable in physiological conditions.
Examples of immunoglobulin scaffolds are the single domain V.sub.H
or V.sub.L scaffold, as well as a single domain camelid V.sub.HH
domain (a form of variable heavy domain found in camelids) or other
immunoglobulin variable domains found in nature or engineered in
the laboratory. In the single domain format disclosed herein, an
immunoglobulin polypeptide need not form a dimer with a second
polypeptide in order to achieve binding activity. Accordingly, any
such polypeptides that naturally contain a cysteine which mediates
disulfide cross-linking to a second protein can be altered to
eliminate the cysteine. Alternatively, the cysteine may be retained
for use in conjugating additional moieties, such as PEG, to the
single domain polypeptide.
[0074] Other scaffolds may be non-antibody scaffold proteins. By
"non-antibody scaffold protein or domain" is meant a non-antibody
polypeptide having an immunoglobulin-like fold. By
"immunoglobulin-like fold" is meant a protein domain of between
about 80-150 amino acid residues that includes two layers of
antiparallel beta-sheets, and in which the flat, hydrophobic faces
of the two beta-sheets are packed against each other. An example of
such a scaffold is the "fibronectin-based scaffold protein", by
which is meant a polypeptide based on a fibronectin type III domain
(Fn3). Fibronectin is a large protein which plays essential roles
in the formation of extracellular matrix and cell-cell
interactions; it consists of many repeats of three types (types I,
II, and III) of small domains (Baron et al., 1991). Fn3 itself is
the paradigm of a large subfamily which includes portions of cell
adhesion molecules, cell surface hormone and cytokine receptors,
chaperoning, and carbohydrate-binding domains for reviews, see Bork
& Doolittle, Proc Natl Acad Sci USA. 1992 Oct. 1;
89(19):8990-4; Bork et al., J Mol Biol. 1994 Sep. 30;
242(4):309-20; Campbell & Spitzfaden, Structure. 1994 May 15;
2(5):333-7; Harpez & Chothia, J Mol Biol. 1994 May 13;
238(4):528-39).
[0075] Preferably, the fibronectin-based scaffold protein is a
".sup.10FN3" scaffold, by which is meant a polypeptide variant
based on the tenth module of the human fibronectin type III protein
in which one or more of the solvent accessible loops has been
randomized or mutated, particularly one or more of the three loops
identified as the BC loop (amino acids 23-30), DE loop (amino acids
52-56) and FG loop (amino acids 77-87) (the numbering scheme is
based on the sequence on the tenth Type III domain of human
fibronectin, with the amino acids Val-Ser-Asp-Val-Pro representing
amino acids numbers 1-5). The amino acid sequence of the wild-type
tenth module of the human fibronectin type III domain is:
VSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKS
TATISGLKPGVDYTITGYAVTGRGDSPASSKPISINYRT (SEQ ID NO:5). Thus, the
wild-type BC loop comprises the sequence of DAPAVTVR; the wild-type
DE loop comprises the sequence of GSKST; the wild-type FG loop
comprises the sequence of GRGDSPASSKP.
[0076] A variety of improved mutant .sup.10Fn3 scaffolds have been
identified. A modified Asp7, which is replaced by a non-negatively
charged amino acid residue (e.g., Asn, Lys, etc.). Both of these
mutations have the effect of promoting greater stability of the
mutant .sup.10Fn3 at neutral pH as compared to the wild-type form.
A variety of additional alterations in the .sup.10Fn3 scaffold that
are either beneficial or neutral have been disclosed. See, for
example, Batori et al. Protein Eng. 2002 December; 15(12):1015-20;
Koide et al., Biochemistry 2001 Aug. 28; 40(34):10326-33.
[0077] Both the variant and wild-type .sup.10Fn3 proteins are
characterized by the same structure, namely seven beta-strand
domain sequences (designated A through and six loop regions (AB
loop, BC loop, CD loop, DE loop, EF loop, and FG loop) which
connect the seven beta-strand domain sequences. The beta strands
positioned closest to the N- and C-termini may adopt a beta-like
conformation in solution. In SEQ ID NO:5, the AB loop corresponds
to residues 15-16, the BC loop corresponds to residues 22-30, the
CD loop corresponds to residues 39-45, the DE loop corresponds to
residues 51-55, the EF loop corresponds to residues 60-66, and the
FG loop corresponds to residues 76-87. The BC loop, DE loop, and FG
loop are all located at the same end of the polypeptide. Similarly,
immunoglobulin scaffolds tend to have at least seven beta or
beta-like strands, and often nine beta or beta-like strands.
Adnectins.TM. can include other Fn3 type fibronectin domains as
long as they exhibit useful activities and properties of .sup.10Fn3
type domains.
VEGFR-2 Binding Proteins
[0078] In preferred embodiments of the invention, the
antiangiogenic polypeptide component comprises a single domain
polypeptide that is a VEGFR-2 specific binder. A single domain
polypeptide disclosed herein may have at least five to seven beta
or beta-like strands distributed between at least two beta sheets,
and at least one loop portion connecting two beta or beta-like
strands, which loop portion participates in binding to VEGFR-2,
particularly KDR, with the binding characterized by a dissociation
constant that is less than 1.times.10.sup.-6M, and preferably less
than 1.times.10.sup.-8M. As described herein, polypeptides having a
dissociation constant of less than 5.times.10.sup.-9M are
particularly desirable for therapeutic use in vivo to inhibit VEGF
signaling. Polypeptides having a dissociation constant of between
1.times.10.sup.-6 M and 5.times.10.sup.-9M may be desirable for use
in detecting or labeling, ex vivo or in vivo, VEGFR-2 proteins.
[0079] Optionally, the VEGFR-2 binding protein will bind
specifically to VEGFR-2 relative to other related proteins from the
same species. By "specifically binds" is meant a polypeptide that
recognizes and interacts with a target protein (e.g., VEGFR-2) but
that does not substantially recognize and interact with other
molecules in a sample, for example, a biological sample. In
preferred embodiments a polypeptide of the invention will
specifically bind a VEGFR with a K.sub.D at least as tight as 500
nM. Preferably, the polypeptide will specifically bind a VEGFR with
a K.sub.D of 1 pM to 500 nM, more preferably 1 pM to 100 nM, more
preferably 1 pM to 10 nM, and most preferably 1 pM to 1 nM or
lower.
[0080] In general, a library of scaffold single domain polypeptides
is screened to identify specific polypeptide variants that can bind
to a chosen target. These libraries may be, for example, phage
display libraries or PROfusion.TM. libraries.
[0081] In an exemplary embodiment, we have exploited a novel in
vitro RNA-protein fusion display technology to isolate polypeptides
that bind to both human (KDR) and mouse (Flk-1) VEGFR-2 and inhibit
VEGF-dependent biological activities. These polypeptides were
identified from libraries of fibronectin-based scaffold proteins
(Koide et al, JMB 284:1141 (1998)) and libraries of V.sub.L domains
in which the diversity of CDR3 has been increased by swapping with
CDR3 domains from a population of V.sub.H molecules. .sup.10Fn3
comprises approximately 94 amino acid residues, as shown in SEQ ID
NO:5.
[0082] In addition, as described above, amino acid sequences at the
N-terminus of .sup.10Fn3 can also be mutated or deleted. For
example, randomization of the BC, DE, and FG loops can occur in the
context of a full-length .sup.10Fn3 or in the context of a
.sup.10Fn3 having a deletion or mutation of 1-8 amino acids of the
N-terminus. For example, the L at position 8 can be mutated to a Q.
After randomization to create a diverse library, fibronectin-based
scaffold proteins can be used in a screening assay to select for
polypeptides with a high affinity for a protein, in this case the
VEGFR. (For a detailed description of the RNA-protein fusion
technology and fibronectin-based scaffold protein library screening
methods see Szostak et al., U.S. Pat. Nos. 6,258,558; 6,261,804;
6,214,553; 6,281,344; 6,207,446; 6,518,018; PCT Publication Numbers
WO 00/34784; WO 01/64942; WO 02/032925; and Roberts and Szostak,
Proc Natl. Acad. Sci. 94:12297-12302, 1997, herein incorporated by
reference.)
[0083] For the initial selection described herein, three regions of
the .sup.10Fn3 at positions 23-29, 52-55 and 77-86 were randomized
and used for in vitro selection against the extracellular domain of
human VEGFR-2 (amino acids 1-764 of KDR fused to human IgG1Fc).
Using mRNA display (RNA-protein fusion) and in vitro selection, we
sampled a .sup.10Fn3-based library with approximately ten trillion
variants. The initial selection identified polypeptides with
moderate affinity (K.sub.D=0-200 nM) that competed with VEGF for
binding to KDR (human VEGFR-2). Subsequently, a single clone
(K.sub.D=11-13 nM) from the initial selection was subjected to
mutagenesis and further selection. This affinity maturation process
yielded new VEGFR binding polypeptides with dissociation constants
between 60 pM to 2 nM. KDR binders are shown in Table 3. In
addition, we also isolated polypeptides that could bind to Flk-1,
the mouse KDR homolog, from mutagenized populations of KDR binders
that initially had no detectable binding affinity to Flk-1,
resulting in the isolation of polypeptides that exhibit dual
specificities to both human and mouse VEGFR-2. These polypeptides
are shown to bind cells that display KDR or Flk-1 extracellular
domains. They also inhibited cell growth in a VEGF-dependent
proliferation assay. Polypeptides that bind to KDR and Flk-1 are
shown in Table 2, while a selection of preferred KDR binders and
KDR/Flk-1 binders are shown in Table 1.
[0084] Using the VEGFR-2 binding polypeptides identified in these
selections we determined FG loop amino acid consensus sequences
required for the binding of the polypeptides to the VEGFR-2. The
sequences are listed as SEQ ID NOs:1-4 below.
[0085] VEGFR-2 binding polypeptides, such as those of SEQ ID
NOs:1-4, may be formulated alone (as isolated peptides), as part of
a .sup.10Fn3 single domain polypeptide, as part of a full-length
fibronectin, (with a full-length amino terminus or a deleted amino
terminus) or a fragment thereof, in the context of an
immunoglobulin (particularly a single domain immunoglobulin), in
the context of another protein having an immunoglobulin-like fold,
or in the context of another, unrelated protein. The polypeptides
can also be formulated as part of a fusion protein with a
heterologous protein that does not itself bind to or contribute in
binding to a VEGFR. In addition, the polypeptides of the invention
can also be fused to nucleic acids. The polypeptides can also be
engineered as monomers, dimers, or multimers.
[0086] Sequences of the Preferred Consensus VEGFR-2 Binding
Peptides:
TABLE-US-00001 SEQ ID NO:1- (L/M)GXN(G/D)(H/R)EL(L/M)TP
[X can be any amino acid; (/) represents alternative amino acid for
the same position]
TABLE-US-00002 SEQ ID NO:2- XERNGRXL(L/M/N)TP
[X can be any amino acid; (/) represents alternative amino acid for
the same position]
TABLE-US-00003 SEQ ID NO:3- (D/E)GXNXRXXIP
[X can be any amino acid; (/) represents alternative amino acid for
the same position]
TABLE-US-00004 SEQ ID NO:4- (D/E)G(R/P)N(G/E)R(S/L)(S/F)IP
[X can be any amino acid; (/) represents alternative amino acid for
the same position]
[0087] Sequences of the Preferred VEGFR-2 Binding .sup.10Fn3
Polypeptides:
TABLE-US-00005 SEQ ID NO:6
EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTAT
ISGLKPGVDYTITGYAVTMGLYGHELLTPISTNYRT SEQ ID NO:7
EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTAT
ISGLKPGVDYTITGYAVTDGENGQFLLVPISINYRT SEQ ID NO:8
EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTAT
ISGLKPGVDYTITGYAVTMGPNDNELLTPISINYRT SEQ ID NO:9
EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTAT
ISGLKPGVDYTITGYAVTAGWDDHELFIPISINYRT SEQ ID NO:10
EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTAT
ISGLKPGVDYTITGYAVTSGHNDHMLMIPISINYRT SEQ ID NO:11
EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTAT
ISGLKPGVDYTITGYAVTAGYNDQILMTPISINYRT SEQ ID NO:12
EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTAT
ISGLKPGVDYTITGYAVTFGLYGKELLIPISINYRT SEQ ID NO:13
EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTAT
ISGLKPGVDYTITGYAVTTGPNDRLLFVPISINYRT SEQ ID NO:14
EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTAT
ISGLKPGVDYTITGYAVTDVYNDHEIKTPISINYRT SEQ ID NO:15
EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTAT
ISGLKPGVDYTITGYAVTDGKDGRVLLTPISINYRT SEQ ID NO:16
EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTAT
ISGLKPGVDYTITGYAVTEVHHDREIKTPISINYRT SEQ ID NO:17
EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTAT
ISGLKPGVDYTITGYAVTQAPNDRVLYTPISINYRT SEQ ID NO:18
EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTAT
ISGLKPGVDYTITGYAVTREENDHELLIPISINYRT SEQ ID NO:19
EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTAT
ISGLKPGVDYTITGYAVTVTHNGHPLMTPISINYRT SEQ ID NO:20
EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTAT
ISGLKPGVDYTITGYAVTLALKGHELLTPISINYRT SEQ ID NO:21
VSDVPRDLEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTV
PLQPPTATISGLKPGVDYTITGYAVTVAQNDHELITPISINYRT SEQ ID NO:22
VSDVPRDL/QEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEF
TVPLQPPAATISGLKPGVDYTITGYAVTMAQSGHELFTPISINYRT SEQ ID NO:24
EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTAT
ISGLKPGVDYTITGYAVTVERNGRVLMTPISINYRT SEQ ID NO:25
EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTAT
ISGLKPGVDYTITGYAVTVERNGRHLMTPISINYRT SEQ ID NO:33
EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTAT
ISGLKPGVDYTITGYAVTLERNGRELMTPISINYRT SEQ ID NO:45
EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTAT
ISGLKPGVDYTITGYAVTEERNGRTLRTPISINYRT SEQ ID NO:53
EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTAT
ISGLKPGVDYTITGYAVTVERNDRVLFTPISINYRT SEQ ID NO:57
EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTAT
ISGLKPGVDYTITGYAVTVERNGRELMTPISINYRT SEQ ID NO:62
EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTAT
ISGLKPGVDYTITGYAVTLERNGRELMVPISINYRT SEQ ID NO:63
EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTAT
ISGLKPGVDYTITGYAVTDGRNDRKLMVPISINYRT SEQ ID NO:68
EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTAT
ISGLKPGVDYTITGYAVTDGQNGRLLNVPISINYRT SEQ ID NO:91
EVVAATPTSLLISWRHHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTA
TISGLKPGVDYTITGYAVTVHWNGRELMTPISINYRT SEQ ID NO:92
EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTAT
ISGLKPGVDYTITGYAVTEEWNGRVLMTPISINYRT SEQ ID NO:93
EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTAT
ISGLKPGVDYTITGYAVTVERNGHTLMTPISINYRT SEQ ID NO:94
EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTAT
ISGLKPGVDYTITGYAVTVEENGRQLMTPISINYRT SEQ ID NO:95
EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTAT
ISGLKPGVDYTITGYAVTLERNGQVLFTPISINYRT SEQ ID NO:96
EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTAT
ISGLKPGVDYTITGYAVTVERNGQVLYTPISINYRT SEQ ID NO:97
EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTAT
ISGLKPGVDYTITGYAVTWGYKDHELLIPISINYRT SEQ ID NO:98
EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTAT
ISGLKPGVDYTITGYAVTLGRNDRELLTPISINYRT SEQ ID NO:99
EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTAT
ISGLKPGVDYTITGYAVTDGPNDRLLNIPISINYRT SEQ ID NO:100
EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTAT
ISGLKPGVDYTITGYAVTFARDGHEILTPISINYRT SEQ ID NO:101
EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTAT
ISGLKPGVDYTITGYAVTLEQNGRELMTPISINYRT SEQ ID NO:102
EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTAT
ISGLKPGVDYTITGYAVTVEENGRVLNTPISINYRT SEQ ID NO:103
EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTAT
ISGLKPGVDYTITGYAVTLEPNGRYLMVPISINYRT SEQ ID NO:104
EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTAT
ISGLKPGVDYTITGYAVTEGRNGRELFIPISINYRT SEQ ID NO:154
VSDVPRDLEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTV
PLQPPAATISGLKPGVDYTITGYAVTWERNGRELFTPISINYRT SEQ ID NO:156
VSDVPRDLEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTV
PLQPPAATISGLKPGVDYTITGYAVTKERNGRELFTPISINYRT SEQ ID NO:172
VSDVPRDLEVVAATPTSLLISWRHPHFPTHYYRITYGETGGNSPVQEFTV
PLQPPAATISGLKPGVDYTITGYAVTTERTGRELFTPISINYRT SEQ ID NO:173
VSDVPRDLEVVAATPTSLLISWRHPHFPTHYYRITYGETGGNSPVQEFTV
PLQPPAATISGLKPGVDYTITGYAVTKERSGRELFTPISINYRT SEQ ID NO:175
VSDVPRDLEVVAATPTSLLISWRHPHFPTHYYRITYGETGGNSPVQEFTV
PLQPPAATISGLKPGVDYTITGYAVTLERDGRELFTPISINYRT SEQ ID NO:177
VSDVPRDLEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTV
PLQPPLATISGLKPGVDYTITG/VYAVTKERNGRELFTPISINYRT SEQ ID NO:180
VSDVPRDLEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTV
PLQPTTATISGLKPGVDYTITGYAVTWERNGRELFTPISINYRT SEQ ID NO:181
VSDVPRDLEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTV
PLQPTVATISGLKPGVDYTITGYAVTLERNDRELFTPISINYRT SEQ ID NO:186
MGEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPT
ATISGLKPGVDYTITVYAVTDGRNGRLLSIPISINYRTEIDKPSQ SEQ ID NO:187
MGEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPT
ATISGLKPGVDYTITVYAVTDGRNGRLLSIPISINYRTEIDKPCQ
SEQ ID NO:188 MVSDVPRDLEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFT
VPLQPPTATISGLKPGVDYTITVYAVTDGRNGRLLSIPISINYRTEIDKPSQ SEQ ID NO:189
MGEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPT
ATISGLKPGVDYTITVYAVTDGWNGRLLSIPISINYRT SEQ ID NO:190
MGEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPT
ATISGLKPGVDYTITVYAVTEGPNERSLFIPISINYRT SEQ ID NO:191
MVSDVPRDLEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFT
VPLQPPTATISGLKPGVDYTITVYAVTEGPNERSLFIPISINYRT SEQ ID NO:192 (A core
form of the polypeptide referred to herein as CT-01):
EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTAT
ISGLKPGVDYTITVYAVTDGRNGRLLSIPISINYRT
[0088] The CT-01 molecule above has a deletion of the first 8 amino
acids and may include additional amino acids at the N- or
C-termini. For example, an additional MG sequence may be placed at
the N-terminus. The M will usually be cleaved off, leaving a GEV .
. . sequence at the N-terminus. The re-addition of the normal 8
amino acids at the N-terminus also produces a KDR binding protein
with desirable properties. The N-terminal methionine is generally
cleaved off to yield a sequence:
TABLE-US-00006 (SEQ ID NO:193)
VSDVPRDLEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTV
PLQPPTATISGLKPGVDYTITVYAVTDGRNGRLLSIPISINYRT.
[0089] A polypeptide disclosed herein may be modified by one or
more conservative substitutions, particularly in portions of the
protein that are not expected to interact with a target protein. It
is expected that as many as 5%, 10%, 20% or even 30% or more of the
amino acids in an immunoglobulin or immunoglobulin-like domain may
be altered by a conservative substitution without substantially
altering the affinity of the protein for target. It may be that
such changes will alter the immunogenicity of the polypeptide in
vivo, and where the immunogenicity is decreased, such changes will
be desirable. As used herein, "conservative substitutions" are
residues that are physically or functionally similar to the
corresponding reference residues. That is, a conservative
substitution and its reference residue have similar size, shape,
electric charge, chemical properties including the ability to form
covalent or hydrogen bonds, or the like. Preferred conservative
substitutions are those fulfilling the criteria defined for an
accepted point mutation in Dayhoff et al., Atlas of Protein
Sequence and Structure 5:345-352 (1978 & Supp.). Examples of
conservative substitutions are substitutions within the following
groups: (a) valine, glycine; (b) glycine, alanine; (c) valine,
isoleucine, leucine; (d) aspartic acid, glutamic acid; (e)
asparagine, glutamine; (f) serine, threonine; (g) lysine, arginine,
methionine; and (h) phenylalanine, tyrosine.
[0090] Polypeptides disclosed herein may also be modified in order
to improve potency, bioavailability, chemical stability, and/or
efficacy. For example, within one embodiment of the invention
D-amino acid peptides, or retroenantio peptide sequences may be
generated in order to improve the bioactivity and chemical
stability of a polypeptide structure (see, e.g., Juvvadi et al., J.
Am. Chem. Soc. 118: 8989-8997, 1996; Freidinger et al., Science,
210: 656-658, 1980). Lactam constraints (see Freidinger, supra),
and/or azabicycloalkane amino acids as dipeptide surrogates can
also be utilized to improve the biological and pharmacological
properties of the native peptides (see, e.g., Hanessian et al.,
Tetrahedron 53:12789-12854, 1997).
[0091] Amide bond surrogates, such as thioamides, secondary and
tertiary amines, heterocycles among others (see review in Spatola,
A. F. in "Chemistry and Biochemistry of Amino Acids, Peptides and
Proteins" Wenstein, B. Ed. Marcel Dekker, New York, 1983 Vol. 7, pp
267-357) can also be utilized to prevent enzymatic degradation of
the polypeptide backbone thereby resulting in improved activity.
Conversion of linear polypeptides to cyclic polypeptide analogs can
also be utilized to improve metabolic stability, since cyclic
polypeptides are much less sensitive to enzymatic degradation (see
generally, Veber, et al. Nature 292:55-58, 1981).
[0092] Polypeptides can also be modified utilizing end group
capping as esters and amides in order to slow or prevent metabolism
and enhance lipophilicity. Dimers of the peptide attached by
various linkers may also enhance activity and specificity (see for
example: Y. Shimohigashi et al, in Peptide Chemistry 1988,
Proceedings of the 26th Symposium on Peptide Chemistry, Tokyo,
October 24-26, pgs. 47-50, 1989). For additional examples of
polypeptide modifications, such as non-natural amino acids, see
U.S. Pat. No. 6,559,126.
[0093] For use in vivo, a form suitable for pegylation may be
generated. For example, a C-terminal tail comprising a cysteine was
added and expressed, as shown below for a CT-01 form lacking the
eight N-terminal amino acids (EIDKPCQ is added at the
C-terminus).
GEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLK
PGVDYTITVYAVTDGRNGRLLSIPISINYRTEIDKPCQ (SEQ ID NO:194). The
pegylated form of this molecule is used in the in vivo experiments
described below. A control form with a serine instead of a cysteine
was also used:
TABLE-US-00007 (SEQ ID NO:195)
GEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTA
TISGLKPGVDYTITVYAVTDGRNGRLLSIPISINYRTEIDKPSQ.
[0094] The same C-terminal tails may also be added to CT-01 forms
having the N-terminal eight amino acids, such as is shown in SEQ ID
NO:193.
[0095] Additional variants with desirable KDR binding properties
were isolated. The following core sequence has a somewhat different
FG loop, and may be expressed with, for example, an N-terminal MG
sequence, an N-terminal sequence that restores the 8 deleted amino
acids, and/or a C-terminal tail to provide a cysteine for
pegylation.
EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKP
GVDYTITVYAVTEGPNERSLFIPISINYRT (SEQ ID NO:196). Another such
variant has the core sequence:
TABLE-US-00008 (SEQ ID NO:197)
VSDVPRDLEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTV
PLQPPTATISGLKPGVDYTITVYAVTEGPNERSLFIPISTNYRT.
[0096] Additionally, preferred single domain immunoglobulin
polypeptides in a V.sub.L framework were isolated by similar
methodology and are disclosed in FIG. 21.
ADDITIONAL PROTEIN EMBODIMENTS
[0097] Proteins of the invention include a single domain
polypeptide as described herein, generally a polypeptide that binds
to a target, such as VEGFR-2, and where target binding activity
situated within a single structural domain, as differentiated from,
for example, antibodies and single chain antibodies, where antigen
binding activity is generally contributed by both a heavy chain
variable domain and a light chain variable domain. The disclosure
also provides larger proteins that may comprise single domain
polypeptides that bind to target. For example, a plurality of
single domain polypeptides may be connected to create a composite
molecule with increased avidity or multivalency. Likewise, a single
domain polypeptide may be attached (e.g., as a fusion protein) to
any number of other polypeptides. In certain aspects a single
domain polypeptide may comprise at least five to seven beta or
beta-like strands distributed among at least two beta sheets, as
exemplified by immunoglobulin and immunoglobulin-like domains. A
beta-like strand is a string of amino acids that participates in
the stabilization of a single domain polypeptide but does not
necessarily adopt a beta strand conformation. Whether a beta-like
strand participates in the stabilization of the protein may be
assessed by deleting the string or altering the sequence of the
string and analyzing whether protein stability is diminished.
Stability may be assessed by, for example, thermal denaturation and
renaturation studies. Preferably, a single domain polypeptide will
include no more than two beta-like strands. A beta-like strand will
not usually adopt an alpha-helical conformation but may adopt a
random coil structure. In the context of an immunoglobulin domain
or an immunoglobulin-like domain, a beta-like strand will most
often occur at the position in the structure that would otherwise
be occupied by the most N-terminal beta strand or the most
C-terminal beta strand. An amino acid string which, if situated in
the interior of a protein sequence would normally form a beta
strand, may, when situated at a position closer to an N- or
C-terminus, adopt a conformation that is not clearly a beta strand
and is referred to herein as a beta-like strand.
[0098] In certain embodiments, the disclosure provides single
domain polypeptides that bind to VEGFR-2. Preferably the single
domain polypeptides bind to human VEGFR-2 or a model species
VEGFR-2. A single domain polypeptide may comprise between about 80
and about 150 amino acids that have a structural organization
comprising: at least seven beta strands or beta-like strands
distributed between at least two beta sheets, and at least one loop
portion connecting two beta strands or beta-like strands, which
loop portion participates in binding to VEGFR-2. In other words a
loop portion may link two beta strands, two beta-like strands or
one beta strand and one beta-like strand. Typically, one or more of
the loop portions will participate in VEGFR-2 binding, although it
is possible that one or more of the beta or beta-like strand
portions will also participate in VEGFR-2 binding, particularly
those beta or beta-like strand portions that are situated closest
to the loop portions. A single domain polypeptide may comprise a
structural unit that is an immunoglobulin domain or an
immunoglobulin-like domain. A single domain polypeptide may bind to
any part of VEGFR-2, although polypeptides that bind to an
extracellular domain of a VEGFR-2 are preferred. Binding may be
assessed in terms of equilibrium constants (e.g., dissociation,
K.sub.D) and in terms of kinetic constants (e.g., on rate constant,
k.sub.on and off rate constant, k.sub.off). A single domain
polypeptide will typically be selected to bind to VEGFR-2 with a
K.sub.D of less than about 10.sup.-6M, or less than about
10.sup.-7M, about 5.times.10.sup.-8M, about 10.sup.-8M or less than
about 10.sup.-9M. VEGFR-2 binding polypeptides may compete for
binding with one, or two or more members of the VEGF family,
particularly VEGF-A, VEGF-C, and/or VEGF-D, and may inhibit one or
more VEGFR-2-mediated biological events, such as proliferation of
cancer cells and cancer metastasis. VEGFR-2 binding polypeptides
may be used for therapeutic purposes as well as for any purpose
involving the detection or binding of VEGFR-2. Polypeptides for
therapeutic use will generally have a K.sub.D of less than
5.times.10.sup.-8M, less than 10.sup.-8M or less than 10.sup.-9M,
although higher K.sub.D values may be tolerated where the k.sub.off
is sufficiently low or the k.sub.on is sufficiently high.
[0099] In certain embodiments, the single domain polypeptide
comprises an immunoglobulin (Ig) variable domain. The Ig variable
domain may, for example, be selected from the group consisting of:
a human V.sub.L domain, a human V.sub.H domain and a camelid
V.sub.HH domain. One, two, three or more loops of the Ig variable
domain may participate in binding to VEGFR-2, and typically any of
the loops known as CDR1, CDR2 or CDR3 will participate in VEGFR-2
binding.
[0100] In certain embodiments, the single domain polypeptide
comprises an immunoglobulin-like domain. One, two, three or more
loops of the immunoglobulin-like domain may participate in binding
to VEGFR-2. A preferred immunoglobulin-like domain is a fibronectin
type III (Fn3) domain. Such domain may comprise, in order from
N-terminus to C-terminus, a beta or beta-like strand, A; a loop,
AB; a beta strand, B; a loop, BC; a beta strand C; a loop CD; a
beta strand D; a loop DE; a beta strand F; a loop FG; and a beta or
beta-like strand G.
[0101] Optionally, any or all of loops AB, BC, CD, DE, EF and FG
may participate in VEGFR-2 binding, although preferred loops are
BC, DE and FG. A preferred Fn3 domain is an Fn3 domain derived from
human fibronectin, particularly the 10.sup.th Fn3 domain of
fibronectin, referred to as .sup.10Fn3. It should be noted that
none of VEGFR-2 binding polypeptides disclosed herein have an amino
acid sequence that is identical to native .sup.10Fn3; the sequence
has been modified to obtain VEGFR-2 binding proteins, but proteins
having the basic structural features of .sup.10Fn3, and
particularly those retaining recognizable sequence homology to the
native .sup.10Fn3 are nonetheless referred to herein as ".sup.10Fn3
polypeptides". This nomenclature is similar to that found in the
antibody field where, for example, a recombinant antibody V.sub.L
domain generated against a particular target protein may not be
identical to any naturally occurring V.sub.L domain but nonetheless
the protein is recognizably a V.sub.L protein. A .sup.10Fn3
polypeptide may be at least 60%, 65%, 70%, 75%, 80%, 85%, or 90%
identical to the human .sup.10Fn3 domain, shown in SEQ ID NO:5.
Much of the variability will generally occur in one or more of the
loops. Each of the beta or beta-like strands of a .sup.10Fn3
polypeptide may consist essentially of an amino acid sequence that
is at least 80%, 85%, 90%, 95% or 100% identical to the sequence of
a corresponding beta or beta-like strand of SEQ ID NO: 5, provided
that such variation does not disrupt the stability of the
polypeptide in physiological conditions. A .sup.10Fn3 polypeptide
may have a sequence in each of the loops AB, CD, and EF that
consists essentially of an amino acid sequence that is at least
80%, 85%, 90%, 95% or 100% identical to the sequence of a
corresponding loop of SEQ ID NO:5. In many instances, any or all of
loops BC, DE, and FG will be poorly conserved relative to SEQ ID
NO:5. For example, all of loops BC, DE, and FG may be less than
20%, 10%, or 0% identical to their corresponding loops in SEQ ID
NO:5.
[0102] In certain embodiments, the disclosure provides a
non-antibody polypeptide comprising a domain having an
immunoglobulin-like fold that binds to VEGFR-2. The non-antibody
polypeptide may have a molecular weight of less than 20 kDa, or
less than 15 kDa and will generally be derived (by, for example,
alteration of the amino acid sequence) from a reference, or
"scaffold", protein, such as an Fn3 scaffold. The non-antibody
polypeptide may bind VEGFR-2 with a K.sub.D less than 10.sup.-6M,
or less than 10.sup.-7M, less than 5.times.10.sup.-8M, less than
10.sup.-8M or less than 10.sup.-9M. The unaltered reference protein
either will not meaningfully bind to VEGFR-2 or will bind with a
K.sub.D of greater than 10.sup.-6M. The non-antibody polypeptide
may inhibit VEGFR-2 signaling, particularly where the non-antibody
polypeptide has a K.sub.D of less than 5.times.10.sup.-8M, less
than 10.sup.-8M or less than 10.sup.-9M, although higher K.sub.D
values may be tolerated where the k.sub.off is sufficiently low
(e.g., less than 5.times.10.sup.-4s.sup.-1). The
immunoglobulin-like fold may be a .sup.10Fn3 polypeptide.
[0103] In certain embodiments, the disclosure provides a
polypeptide comprising a single domain having an immunoglobulin
fold that binds to VEGFR-2. The polypeptide may have a molecular
weight of less than 20 kDa, or less than 15 kDa and will generally
be derived (by, for example, alteration of the amino acid sequence)
from a variable domain of an immunoglobulin. The polypeptide may
bind VEGFR-2 with a K.sub.D less than 10.sup.-6 M, or less than
10.sup.-7M, less than 5.times.10.sup.-8M, less than 10.sup.-8M or
less than 10.sup.-9M. The polypeptide may inhibit VEGFR-2
signaling, particularly where the polypeptide has a K.sub.D of less
than 5.times.10.sup.-8M, less than 10.sup.-8M or less than
10.sup.-9M, although higher K.sub.D values may be tolerated where
the k.sub.off is sufficiently low or where the k.sub.on is
sufficiently high. In some embodiments, the polypeptide comprises
an amino acid sequence that is at least 80% identical to SEQ NO: 5.
In some embodiments, the polypeptide comprises an amino acid
sequence selected from the group consisting of any of SEQ ID NOs:
6-183, 186-197, 199 and 241-310. In some embodiments, the
polypeptide further comprises PEG.
[0104] In certain aspects, the disclosure provides
sustained-release delivery systems that deliver short peptide
sequences that mediate VEGFR-2 binding. Such sequences may mediate
VEGFR-2 binding in an isolated form or when inserted into a
particular protein structure, such as an immunoglobulin or
immunoglobulin-like domain. Examples of such sequences include
those disclosed (such as SEQ ID NOs: 6-183, 186-197, 199 and
241-310) and other sequences that are at least 85%, 90%, or 95%
identical to SEQ ID NO:5 to such sequences and retain VEGFR-2
binding activity. Accordingly, the disclosure provides
substantially pure polypeptides comprising an amino acid sequence
that is at least 85% identical to the sequence of any of such
sequences, wherein said polypeptide binds to a VEGFR-2 and competes
with an VEGF species for binding to VEGFR-2. Examples of such
polypeptides include a polypeptide comprising an amino acid
sequence that is at least 80%, 85%, 90%, 95% or 100% identical to
an amino acid sequence of SEQ ID: 6-183, 186-197, 199 and 241-310.
Preferably such polypeptides will inhibit a biological activity of
a VEGF and may bind to VEGFR-2 with a K.sub.D less than 10.sup.-6
M, or less than 10.sup.-7M, less than 5.times.10.sup.-8M, less than
10.sup.-8M or less than 10.sup.-9M.
[0105] In certain embodiments, any of the VEGFR-2 binding
polypeptides described herein may be bound to one or more
additional moieties, including, for example, a moiety that also
binds to VEGFR-2 (e.g., a second identical or different VEGFR-2
binding polypeptide), a moiety that binds to a different target
(e.g., to create a dual-specificity binding agent), a labeling
moiety, a moiety that facilitates protein purification or a moiety
that provides improved pharmacokinetics. Improved pharmacokinetics
may be assessed according to the perceived therapeutic need. Often
it is desirable to increase bioavailability and/or increase the
time between doses, possibly by increasing the time that a protein
remains available in the serum after dosing. In some instances, it
is desirable to improve the continuity of the serum concentration
of the protein over time (e.g., decrease the difference in serum
concentration of the protein shortly after administration and
shortly before the next administration). Moieties that tend to slow
clearance of a protein from the blood include polyethylene glycol,
sugars (e.g. sialic acid), and well-tolerated protein moieties
(e.g., Fc fragment or serum albumin). The single domain polypeptide
may be attached to a moiety that reduces the clearance rate of the
polypeptide in a mammal (e.g., mouse, rat, or human) by greater
than three-fold relative to the unmodified polypeptide. Other
measures of improved pharmacokinetics may include serum half-life,
which is often divided into an alpha phase and a beta phase. Either
or both phases may be improved significantly by addition of an
appropriate moiety. Where polyethylene glycol is employed, one or
more PEG molecules may be attached at different positions in the
protein, and such attachment may be achieved by reaction with
amines, thiols or other suitable reactive groups. Pegylation may be
achieved by site-directed pegylation, wherein a suitable reactive
group is introduced into the protein to create a site where
pegylation preferentially occurs. In a preferred embodiment, the
protein is modified so as to have a cysteine residue at a desired
position, permitting site directed pegylation on the cysteine. PEG
may vary widely in molecular weight and may be branched or linear.
Notably, the present disclosure establishes that pegylation is
compatible with target binding activity of .sup.10Fn3 polypeptides
and, further, that pegylation does improve the pharmacokinetics of
such polypeptides. Accordingly, in one embodiment, the disclosure
provides pegylated forms of .sup.10Fn3 polypeptides, regardless of
the target that can be bound by such polypeptides.
Nucleic Acids and Production of Polypeptides
[0106] Polypeptides of the present invention can be produced using
any standard methods known in the art. In one example, the
polypeptides are produced by recombinant DNA methods by inserting a
nucleic acid sequence (e.g., a cDNA) encoding the polypeptide into
a recombinant expression vector and expressing the DNA sequence
under conditions promoting expression.
[0107] Nucleic acids encoding any of the various polypeptides
disclosed herein may be synthesized chemically. Codon usage may be
selected so as to improve expression in a cell. Such codon usage
will depend on the cell type selected. Specialized codon usage
patterns have been developed for E. coli and other bacteria, as
well as mammalian cells, plant cells, yeast cells and insect cells.
See for example: Mayfield et al., Proc Natl Acad Sci USA. 2003 Jan.
21; 100(2):438-42; Sinclair et al. Protein Expr Purif. 2002
October; 26(1):96-105; Connell N D. Curr Opin Biotechnol. 2001
October; 12(5):446-9; Makrides et al. Microbiol. Rev. 1996
September; 60(3):512-38; and Sharp et al. Yeast. 1991 October;
7(7):657-78.
[0108] Examples of nucleic acid sequences encoding a CT-01
polypeptide disclosed herein are:
TABLE-US-00009 SEQ ID NO:184
atgggcgaagttgttgctgcgacccccaccagcctactgatcagctggcg
ccacccgcacttcccgactagatattacaggatcacttacggagaaacag
gaggaaatagccctgtccaggagttcactgtgcctctgcagccccccaca
gctaccatcagcggccttaaacctggagttgattataccatcactgtgta
tgctgtcactgacggccggaacgggcgcctcctgagcatcccaatttcca
ttaattaccgcacagaaattgacaaaccatgccag SEQ ID NO:185
atgggcgaagttgttgctgcgacccccaccagcctactgatcagctggcg
ccacccgcacttcccgactagatattacaggatcacttacggagaaacag
gaggaaatagccctgtccaggagttcactgtgcctctgcagccccccaca
gctaccatcagcggccttaaacctggagttgattataccatcactgtgta
tgctgtcactgacggccggaacgggcgcctcctgagcatcccaatttcca
ttaattaccgcaca
[0109] General techniques for nucleic acid manipulation are
described for example in Sambrook et al., Molecular Cloning: A
Laboratory Manual, Vols. 1-3, Cold Spring Harbor Laboratory Press,
2 ed., 1989, or F. Ausubel et al., Current Protocols in Molecular
Biology (Green Publishing and Wiley-Interscience: New York, 1987)
and periodic updates, herein incorporated by reference. The DNA
encoding the polypeptide is operably linked to suitable
transcriptional or translational regulatory elements derived from
mammalian, viral, or insect genes. Such regulatory elements include
a transcriptional promoter, an optional operator sequence to
control transcription, a sequence encoding suitable mRNA ribosomal
binding sites, and sequences that control the termination of
transcription and translation. The ability to replicate in a host,
usually conferred by an origin of replication, and a selection gene
to facilitate recognition of transformants are additionally
incorporated.
[0110] The recombinant DNA can also include any type of protein tag
sequence that may be useful for purifying the protein. Examples of
protein tags include but are not limited to a histidine tag, a FLAG
tag, a myc tag, an HA tag, or a GST tag. Appropriate cloning and
expression vectors for use with bacterial, fungal, yeast, and
mammalian cellular hosts can be found in Cloning Vectors: A
Laboratory Manual, (Elsevier, New York, 1985), the relevant
disclosure of which is hereby incorporated by reference.
[0111] The expression construct is introduced into the host cell
using a method appropriate to the host cell, as will be apparent to
one of skill in the art. A variety of methods for introducing
nucleic acids into host cells are known in the art, including, but
not limited to, electroporation; transfection employing calcium
chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or
other substances; microprojectile bombardment; lipofection; and
infection (where the vector is an infectious agent).
[0112] Suitable host cells include prokaryotes, yeast, mammalian
cells, or bacterial cells. Suitable bacteria include gram negative
or gram positive organisms, for example, E. coli or Bacillus spp.
Yeast, preferably from the Saccharomyces species, such as S.
cerevisiae, may also be used for production of polypeptides.
Various mammalian or insect cell culture systems can also be
employed to express recombinant proteins. Baculovirus systems for
production of heterologous proteins in insect cells are reviewed by
Luckow and Summers, (Bio/Technology, 6:47, 1988). Examples of
suitable mammalian host cell lines include endothelial cells, COS-7
monkey kidney cells, CV-1, L cells, C127, 3T3, Chinese hamster
ovary (CHO), human embryonic kidney cells, HeLa, 293, 293T, and BHK
cell lines. Purified polypeptides are prepared by culturing
suitable host/vector systems to express the recombinant proteins.
For many applications, the small size of many of the polypeptides
disclosed herein would make expression in E. coli as the preferred
method for expression. The protein is then purified from culture
media or cell extracts.
[0113] Proteins disclosed herein can also be produced using
cell-translation systems. For such purposes the nucleic acids
encoding the polypeptide must be modified to allow in vitro
transcription to produce mRNA and to allow cell-free translation of
the mRNA in the particular cell-free system being utilized
(eukaryotic such as a mammalian or yeast cell-free translation
system or prokaryotic such as a bacterial cell-free translation
system.
[0114] VEGFR-binding polypeptides can also be produced by chemical
synthesis (e.g., by the methods described in Solid Phase Peptide
Synthesis, 2nd ed., 1984, The Pierce Chemical Co., Rockford, Ill.).
Modifications to the protein can also be produced by chemical
synthesis.
[0115] The polypeptide of the present invention can be purified by
isolation/purification methods for proteins generally known in the
field of protein chemistry. Non-limiting examples include
extraction, recrystallization, salting out (e.g., with ammonium
sulfate or sodium sulfate), centrifugation, dialysis,
ultrafiltration, adsorption chromatography, ion exchange
chromatography, hydrophobic chromatography, normal phase
chromatography, reversed-phase chromatography, gel filtration, gel
permeation chromatography, affinity chromatography,
electrophoresis, countercurrent distribution or any combinations of
these. After purification, polypeptides may be exchanged into
different buffers and/or concentrated by any of a variety of
methods known to the art, including, but not limited to, filtration
and dialysis.
[0116] The purified polypeptide is preferably at least 85% pure,
more preferably at least 95% pure, and most preferably at least 98%
pure. Regardless of the exact numerical value of the purity, the
polypeptide is sufficiently pure for use as a pharmaceutical
product. The polypeptide is in particular free of endotoxins
Post-Translational Modifications of Polypeptides
[0117] In certain embodiments, the binding polypeptides of the
invention may further comprise post-translational modifications.
Exemplary post-translational protein modification include
phosphorylation, acetylation, methylation, ADP-ribosylation,
ubiquitination, glycosylation, carbonylation, sumoylation,
biotinylation or addition of a polypeptide side chain or of a
hydrophobic group. As a result, the modified soluble polypeptides
may contain non-amino acid elements, such as lipids, poly- or
mono-saccharide, and phosphates. A preferred form of glycosylation
is sialylation, which conjugates one or more sialic acid moieties
to the polypeptide. Sialic acid moieties improve solubility and
serum half-life while also reducing the possible immunogeneticity
of the protein. See, e.g., Raju et al. Biochemistry. 2001 Jul. 31;
40(30):8868-76. Effects of such non-amino acid elements on the
functionality of a polypeptide may be tested for its antagonizing
role in VEGFR-2 or VEGF function, e.g., its inhibitory effect on
angiogenesis or on tumor growth.
[0118] In one specific embodiment of the present invention,
modified forms of the subject soluble polypeptides comprise linking
the subject soluble polypeptides to nonproteinaceous polymers. In
one specific embodiment, the polymer is polyethylene glycol
("PEG"), polypropylene glycol, or polyoxyalkylenes, in the manner
as set forth in U.S. Pat. No. 4,640,835; 4,496,689; 4,301,144;
4,670,417; 4,791,192 or 4,179,337. Examples of the modified
polypeptide of the invention include PEGylated CT-322.
[0119] PEG is a well-known, water soluble polymer that is
commercially available or can be prepared by ring-opening
polymerization of ethylene glycol according to methods well known
in the art (Sandler and Karo, Polymer Synthesis, Academic Press,
New York, Vol. 3, pages 138-161). The term "PEG" is used broadly to
encompass any polyethylene glycol molecule, without regard to size
or to modification at an end of the PEG, and can be represented by
the formula:
X--O(CH.sub.2CH.sub.2O).sub.n-1CH.sub.2CH.sub.2OH (1),
where n is 20 to 2300 and X is H or a terminal modification, e.g.,
a C.sub.1-4 alkyl. In one embodiment, the PEG of the invention
terminates on one end with hydroxy or methoxy, i.e., X is H or
CH.sub.3 ("methoxy PEG"). A PEG can contain further chemical groups
which are necessary for binding reactions; which results from the
chemical synthesis of the molecule; or which is a spacer for
optimal distance of parts of the molecule. In addition, such a PEG
can consist of one or more PEG side-chains which are linked
together. PEGs with more than one PEG chain are called multiarmed
or branched PEGs. Branched PEGs can be prepared, for example, by
the addition of polyethylene oxide to various polyols, including
glycerol, pentaerythriol, and sorbitol. For example, a four-armed
branched PEG can be prepared from pentaerythriol and ethylene
oxide. Branched PEG are described in, for example, EP-A 0 473 084
and U.S. Pat. No. 5,932,462. One form of PEGs includes two PEG
side-chains (PEG2) linked via the primary amino groups of a lysine
(Monfardini, C., et al., Bioconjugate Chem. 6 (1995) 62-69).
[0120] In a preferred embodiment, the pegylated .sup.10Fn3
polypeptide is produced by site-directed pegylation, particularly
by conjugation of PEG to a cysteine moiety at the N- or C-terminus.
Accordingly, the present disclosure provides a target-binding
.sup.10Fn3 polypeptide with improved pharmacokinetic properties,
the polypeptide comprising: a .sup.10Fn3 domain having from about
80 to about 150 amino acids, wherein at least one of the loops of
said .sup.10Fn3 domain participate in target binding; and a
covalently bound PEG moiety, wherein said .sup.10Fn3 polypeptide
binds to the target with a K.sub.D of less than 100 nM and has a
clearance rate of less than 30 mL/hr/kg in a mammal. The PEG moiety
may be attached to the .sup.10Fn3 polypeptide by site directed
pegylation, such as by attachment to a Cys residue, where the Cys
residue may be positioned at the N-terminus of the .sup.10Fn3
polypeptide or between the N-terminus and the most N-terminal beta
or beta-like strand or at the C-terminus of the .sup.10Fn3
polypeptide or between the C-terminus and the most C-terminal beta
or beta-like strand. A Cys residue may be situated at other
positions as well, particularly any of the loops that do not
participate in target binding. A PEG moiety may also be attached by
other chemistry, including by conjugation to amines.
[0121] PEG conjugation to peptides or proteins generally involves
the activation of PEG and coupling of the activated
PEG-intermediates directly to target proteins/peptides or to a
linker, which is subsequently activated and coupled to target
proteins/peptides (see Abuchowski, A. et al, J. Biol. Chem., 252,
3571 (1977) and J. Biol. Chem., 252, 3582 (1977), Zalipsky, et al.,
and Harris et. al., in: Poly(ethylene glycol) Chemistry:
Biotechnical and Biomedical Applications; (J. M. Harris ed.) Plenum
Press: New York, 1992; Chap. 21 and 22). It is noted that a binding
polypeptide containing a PEG molecule is also known as a conjugated
protein, whereas the protein lacking an attached PEG molecule can
be referred to as unconjugated.
[0122] A variety of molecular mass forms of PEG can be selected,
e.g., from about 1,000 Daltons (Da) to 100,000 Da (n is 20 to
2300), for conjugating to VEGFR-2 binding polypeptides. The number
of repeating units "n" in the PEG is approximated for the molecular
mass described in Daltons. It is preferred that the combined
molecular mass of PEG on an activated linker is suitable for
pharmaceutical use. Thus, in one embodiment, the molecular mass of
the PEG molecules does not exceed 100,000 Da. For example, if three
PEG molecules are attached to a linker, where each PEG molecule has
the same molecular mass of 12,000 Da (each n is about 270), then
the total molecular mass of PEG on the linker is about 36,000 Da
(total n is about 820). The molecular masses of the PEG attached to
the linker can also be different, e.g., of three molecules on a
linker two PEG molecules can be 5,000 Da each (each n is about 110)
and one PEG molecule can be 12,000 Da (n is about 270).
[0123] In a specific embodiment of the invention, a VEGFR-2 binding
polypeptide is covalently linked to one poly(ethylene glycol) group
of the formula:
--CO--(CH.sub.2).sub.x--(OCH.sub.2CH.sub.2).sub.m--OR, with the
--CO (i.e. carbonyl) of the poly(ethylene glycol) group forming an
amide bond with one of the amino groups of the binding polypeptide;
R being lower alkyl; x being 2 or 3; m being from about 450 to
about 950; and n and m being chosen so that the molecular weight of
the conjugate minus the binding polypeptide is from about 10 to 40
kDa. In one embodiment, an binding polypeptide's .epsilon.-amino
group of a lysine is the available (free) amino group.
[0124] The above conjugates may be more specifically presented by
formula (II):
P--NHCO--(CH.sub.2).sub.x--(OCH.sub.2CH.sub.2).sub.m--OR (II),
wherein P is the group of a binding polypeptide as described
herein, (i.e. without the amino group or amino groups which form an
amide linkage with the carbonyl shown in formula (II); and wherein
R is lower alkyl; x is 2 or 3; m is from about 450 to about 950 and
is chosen so that the molecular weight of the conjugate minus the
binding polypeptide is from about 10 to about 40 kDa. As used
herein, the given ranges of "m" have an orientational meaning. The
ranges of "m" are determined in any case, and exactly, by the
molecular weight of the PEG group.
[0125] One skilled in the art can select a suitable molecular mass
for PEG, e.g., based on how the pegylated binding polypeptide will
be used therapeutically, the desired dosage, circulation time,
resistance to proteolysis, immunogenicity, and other
considerations. For a discussion of PEG and its use to enhance the
properties of proteins, see N. V. Katre, Advanced Drug Delivery
Reviews 10: 91-114 (1993).
[0126] In one embodiment of the invention, PEG molecules may be
activated to react with amino groups on a binding polypeptide, such
as with lysines (Bencham C. O. et al., Anal. Biochem., 131, 25
(1983); Veronese, F. M. et al., Appl. Biochem., 11, 141 (1985).;
Zalipsky, S. et al., Polymeric Drugs and Drug Delivery Systems,
adrs 9-110 ACS Symposium Series 469 (1999); Zalipsky, S. et al.,
Europ. Polym. J., 19, 1177-1183 (1983); Delgado, C. et al.,
Biotechnology and Applied Biochemistry, 12, 119-128 (1990)).
[0127] In one specific embodiment, carbonate esters of PEG are used
to form the PEG-binding polypeptide conjugates.
N,N'-disuccinimidylcarbonate (DSC) may be used in the reaction with
PEG to form active mixed PEG-succinimidyl carbonate that may be
subsequently reacted with a nucleophilic group of a linker or an
amino group of a binding polypeptide (see U.S. Pat. No. 5,281,698
and U.S. Pat. No. 5,932,462). In a similar type of reaction,
1,1'-(dibenzotriazolyl)carbonate and di-(2-pyridyl)carbonate may be
reacted with PEG to form PEG-benzotriazolyl and PEG-pyridyl mixed
carbonate (U.S. Pat. No. 5,382,657), respectively.
[0128] Pegylation of a .sup.10Fn3 polypeptide can be performed
according to the methods of the state of the art, for example by
reaction of the binding polypeptide with electrophilically active
PEGs (supplier: Shearwater Corp., USA, www.shearwatercorp.com).
Preferred PEG reagents of the present invention are, e.g.,
N-hydroxysuccinimidyl propionates (PEG-SPA), butanoates (PEG-SBA),
PEG-succinimidyl propionate or branched N-hydroxysuccinimides such
as mPEG2--NHS (Monfardini, C., et al., Bioconjugate Chem. 6 (1995)
62-69). Such methods may used to pegylated at an E-amino group of a
binding polypeptide lysine or the N-terminal amino group of the
binding polypeptide.
[0129] In another embodiment, PEG molecules may be coupled to
sulfhydryl groups on a binding polypeptide (Sartore, L., et al.,
Appl. Biochem. Biotechnol., 27, 45 (1991); Morpurgo et al., Biocon.
Chem., 7, 363-368 (1996); Goodson et al., Bio/Technology (1990) 8,
343; U.S. Pat. No. 5,766,897). U.S. Pat. Nos. 6,610,281 and
5,766,897 describes exemplary reactive PEG species that may be
coupled to sulfhydryl groups.
[0130] In some embodiments where PEG molecules are conjugated to
cysteine residues on a binding polypeptide, the cysteine residues
are native to the binding polypeptide, whereas in other
embodiments, one or more cysteine residues are engineered into the
binding polypeptide. Mutations may be introduced into an binding
polypeptide coding sequence to generate cysteine residues. This
might be achieved, for example, by mutating one or more amino acid
residues to cysteine. Preferred amino acids for mutating to a
cysteine residue include serine, threonine, alanine and other
hydrophilic residues. Preferably, the residue to be mutated to
cysteine is a surface-exposed residue. Algorithms are well-known in
the art for predicting surface accessibility of residues based on
primary sequence or a protein. Alternatively, surface residues may
be predicted by comparing the amino acid sequences of binding
polypeptides, given that the crystal structure of the framework
based on which binding polypeptides are designed and evolved has
been solved (see Himanen et al., Nature. (2001) 20-27;
414(6866):933-8) and thus the surface-exposed residues identified.
In one embodiment, cysteine residues are introduced into binding
polypeptides at or near the N- and/or C-terminus, or within loop
regions.
[0131] In some embodiments, the pegylated binding polypeptide
comprises a PEG molecule covalently attached to the alpha amino
group of the N-terminal amino acid. Site specific N-terminal
reductive amination is described in Pepinsky et al., (2001) JPET,
297, 1059, and U.S. Pat. No. 5,824,784. The use of a PEG-aldehyde
for the reductive amination of a protein utilizing other available
nucleophilic amino groups is described in U.S. Pat. No. 4,002,531,
in Wieder et al., (1979) J. Biol. Chem. 254, 12579, and in Chamow
et al., (1994) Bioconjugate Chem. 5, 133.
[0132] In another embodiment, pegylated binding polypeptide
comprises one or more PEG molecules covalently attached to a
linker, which in turn is attached to the alpha amino group of the
amino acid residue at the N-terminus of the binding polypeptide.
Such an approach is disclosed in U.S. Patent Publication No.
2002/0044921 and in WO94/01451.
[0133] In one embodiment, a binding polypeptide is pegylated at the
C-terminus. In a specific embodiment, a protein is pegylated at the
C-terminus by the introduction of C-terminal azido-methionine and
the subsequent conjugation of a methyl-PEG-triarylphosphine
compound via the Staudinger reaction. This C-terminal conjugation
method is described in Cazalis et al., C-Terminal Site-Specific
PEGylation of a Truncated Thrombomodulin Mutant with Retention of
Full Bioactivity, Bioconjug Chem. 2004; 15(5): 1005-1009.
[0134] Monopegylation of a binding polypeptide can also be produced
according to the general methods described in WO 94/01451. WO
94/01451 describes a method for preparing a recombinant polypeptide
with a modified terminal amino acid alpha-carbon reactive group.
The steps of the method involve forming the recombinant polypeptide
and protecting it with one or more biologically added protecting
groups at the N-terminal alpha-amine and C-terminal alpha-carboxyl.
The polypeptide can then be reacted with chemical protecting agents
to selectively protect reactive side chain groups and thereby
prevent side chain groups from being modified. The polypeptide is
then cleaved with a cleavage reagent specific for the biological
protecting group to form an unprotected terminal amino acid
alpha-carbon reactive group. The unprotected terminal amino acid
alpha-carbon reactive group is modified with a chemical modifying
agent. The side chain protected terminally modified single copy
polypeptide is then deprotected at the side chain groups to form a
terminally modified recombinant single copy polypeptide. The number
and sequence of steps in the method can be varied to achieve
selective modification at the N- and/or C-terminal amino acid of
the polypeptide.
[0135] The ratio of a binding polypeptide to activated PEG in the
conjugation reaction can be from about 1:0.5 to 1:50, between from
about 1:1 to 1:30, or from about 1:5 to 1:15. Various aqueous
buffers can be used in the present method to catalyze the covalent
addition of PEG to the binding polypeptide. In one embodiment, the
pH of a buffer used is from about 7.0 to 9.0. In another
embodiment, the pH is in a slightly basic range, e.g., from about
7.5 to 8.5. Buffers having a pKa close to neutral pH range may be
used, e.g., phosphate buffer.
[0136] Conventional separation and purification techniques known in
the art can be used to purify PEGylated binding polypeptide, such
as size exclusion (e.g. gel filtration) and ion exchange
chromatography. Products may also be separated using SDS-PAGE.
Products that may be separated include mono-, di-, tri- poly- and
un-pegylated binding polypeptide, as well as free PEG. The
percentage of mono-PEG conjugates can be controlled by pooling
broader fractions around the elution peak to increase the
percentage of mono-PEG in the composition. About ninety percent
mono-PEG conjugates represents a good balance of yield and
activity. Compositions in which, for example, at least ninety-two
percent or at least ninety-six percent of the conjugates are
mono-PEG species may be desired. In an embodiment of this invention
the percentage of mono-PEG conjugates is from ninety percent to
ninety-six percent.
[0137] In one embodiment, PEGylated binding polypeptide of the
invention contain one, two or more PEG moieties. In one embodiment,
the PEG moiety(ies) are bound to an amino acid residue which is on
the surface of the protein and/or away from the surface that
contacts the target ligand. In one embodiment, the combined or
total molecular mass of PEG in PEG-binding polypeptide is from
about 3,000 Da to 60,000 Da, optionally from about 10,000 Da to
36,000 Da. In a one embodiment, the PEG in pegylated binding
polypeptide is a substantially linear, straight-chain PEG.
[0138] In one embodiment of the invention, the PEG in pegylated
binding polypeptide is not hydrolyzed from the pegylated amino acid
residue using a hydroxylamine assay, e.g., 450 mM hydroxylamine (pH
6.5) over 8 to 16 hours at room temperature, and is thus stable. In
one embodiment, greater than 80% of the composition is stable
mono-PEG-binding polypeptide, more preferably at least 90%, and
most preferably at least 95%.
[0139] In another embodiment, the pegylated binding polypeptides of
the invention will preferably retain at least 25%, 50%, 60%, 70%
least 80%, 85%, 90%, 95% or 100% of the biological activity
associated with the unmodified protein. In one embodiment,
biological activity refers to its ability to bind to VEGFR-2, as
assessed by K.sub.D, k.sub.on, or k.sub.off. In one specific
embodiment, the pegylated binding polypeptide protein shows an
increase in binding to VEGFR relative to unpegylated binding
polypeptide.
[0140] The serum clearance rate of PEG-modified polypeptide may be
decreased by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or even
90%, relative to the clearance rate of the unmodified binding
polypeptide. The PEG-modified polypeptide may have a half-life
(t.sub.1/2) which is enhanced relative to the half-life of the
unmodified protein. The half-life of PEG-binding polypeptide may be
enhanced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,
100%, 125%, 150%, 175%, 200%, 250%, 300%, 400% or 500%, or even by
1000% relative to the half-life of the unmodified binding
polypeptide. In some embodiments, the protein half-life is
determined in vitro, such as in a buffered saline solution or in
serum. In other embodiments, the protein half-life is an in vivo
half life, such as the half-life of the protein in the serum or
other bodily fluid of an animal.
Therapeutic Formulations and Modes of Administration
[0141] The present invention provides sustained-release intraocular
drug delivery systems that are useful, in particular, for
inhibiting VEGF biological activity. Techniques and dosages for
administration vary depending on the type of specific polypeptide
and the specific condition being treated but can be readily
determined by the skilled artisan. In general, regulatory agencies
require that a protein reagent to be used as a therapeutic be
formulated so as to have acceptably low levels of pyrogens.
Accordingly, therapeutic formulations will generally be
distinguished from other formulations in that they are
substantially pyrogen free, or at least contain no more than
acceptable levels of pyrogen as determined by the appropriate
regulatory agency (e.g., FDA). A pyrogen may be an endotoxin or
exotoxin. In some embodiments, the drug delivery system is
substantially endotoxin free.
[0142] Therapeutic compositions of the present invention may be
administered with a pharmaceutically acceptable diluent, carrier,
or excipient, in unit dosage form. Methods well known in the art
for making formulations are found, for example, in "Remington: The
Science and Practice of Pharmacy" (20th ed., ed. A. R. Gennaro A
R., 2000, Lippincott Williams & Wilkins, Philadelphia, Pa.).
Formulations for parenteral administration may, for example,
contain excipients, sterile water, saline, polyalkylene glycols
such as polyethylene glycol, oils of vegetable origin, or
hydrogenated napthalenes. Biocompatible, biodegradable lactide
polymer, lactide/glycolide copolymer, or
polyoxyethylene-polyoxypropylene copolymers may be used to control
the release of the compounds. Nanoparticulate formulations (e.g.,
biodegradable nanoparticles, solid lipid nanoparticles, liposomes)
may be used to control the biodistribution of the compounds. Other
potentially useful parenteral delivery systems include
ethylene-vinyl acetate copolymer particles, osmotic pumps,
implantable infusion systems, and liposomes. The concentration of
the compound in the formulation varies depending upon a number of
factors, including the dosage of the drug to be administered, and
the route of administration.
[0143] The antiangiogenic polypeptide may be optionally
administered as a pharmaceutically acceptable salt, such as
non-toxic acid addition salts or metal complexes that are commonly
used in the pharmaceutical industry. Examples of acid addition
salts include organic acids such as acetic, lactic, pamoic, maleic,
citric, malic, ascorbic, succinic, benzoic, palmitic, suberic,
salicylic, tartaric, methanesulfonic, toluenesulfonic, or
trifluoroacetic acids or the like; polymeric acids such as tannic
acid, carboxymethyl cellulose, or the like; and inorganic acid such
as hydrochloric acid, hydrobromic acid, sulfuric acid phosphoric
acid, or the like. Metal complexes include zinc, iron, and the
like. In one example, the polypeptide is formulated in the presence
of sodium acetate to increase thermal stability.
[0144] The antiangiogenic polypeptides may also be entrapped in
microcapsule prepared, for example, by coacervation techniques or
by interfacial polymerization, for example, hydroxymethylcellulose
or gelatin-microcapsule and poly-(methylmethacylate) microcapsule,
respectively, in colloidal drug delivery systems (for example,
liposomes, albumin microspheres, microemulsions, nano-particles and
nanocapsules) or in macroemulsions. Such techniques are disclosed
in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed.
(1980).
[0145] The formulations to be used for in vivo administration must
be sterile. This is readily accomplished by filtration through
sterile filtration membranes.
[0146] A therapeutically effective dose refers to a dose that
produces the therapeutic effects for which it is administered. The
exact dose will depend on the disorder to be treated, and may be
ascertained by one skilled in the art using known techniques. In
addition, as is known in the art, adjustments for age as well as
the body weight, general health, sex, diet, time of administration,
drug interaction, and the severity of the disease may be necessary,
and will be ascertainable with routine experimentation by those
skilled in the art.
[0147] In some embodiments, the sustained-release drug delivery
system is a liquid or a gel composition, suitable for injection
into the ocular region of a patient. In some embodiments, the
sustained release drug delivery system is a biodegradable implant.
The drug system is injected intraocularly, such as an intravitreal,
subconjunctival injection, or subtenon injection; and the resulting
implant releases drug over a predetermined interval of time.
Typically, the implant biodegrades at the same rate that the drug
is released; therefore, the injection site essentially resolves in
time for the next injection.
[0148] Sustained-release drug delivery systems also include
semipermeable matrices of solid hydrophobic polymers containing the
proteins of the invention, which matrices are in the form of shaped
articles, e.g., films, or microcapsule. Examples of
sustained-release matrices include polyesters, hydrogels (for
example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),
polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic
acid and y ethyl-L-glutamate, non-degradable ethylene-vinyl
acetate, degradable lactic acid-glycolic acid copolymers such as
the LUPRON DEPOT.TM. (injectable microspheres composed of lactic
acid-glycolic acid copolymer and leuprolide acetate), and
poly-D-(-)-3-hydroxybutyric acid. While polymers such as
ethylene-vinyl acetate and lactic acid-glycolic acid enable release
of molecules for over 100 days, certain hydrogels release proteins
for shorter time periods. When encapsulated proteins of the
invention may remain in the body for a long time, they may denature
or aggregate as a result of exposure to moisture at 37.degree. C.,
resulting in a loss of biological activity and possible changes in
immunogenicity. Rational strategies can be devised for
stabilization depending on the mechanism involved. For example, if
the aggregation mechanism is discovered to be intermolecular S--S
bond formation through thio-disulfide interchange, stabilization
may be achieved by modifying sulfhydryl residues, lyophilizing from
acidic solutions, controlling moisture content, using appropriate
additives, and developing specific polymer matrix compositions.
[0149] In some embodiments, the sustained release drug delivery
system utilizes the Atrigel.TM. system comprising lactide/glycolide
copolymers as described in U.S. Patent Application 20060210604. In
some embodiments, the sustained release drug delivery system
utilizes a biodegradable PLGA intravitreal implant as described in
U.S. Patent Application 20050244469.
[0150] Other drug delivery systems have been previously described
and may be used to deliver the antiangiogenic polypeptide
component. The following is a list of suitable implants that may be
used in the drug delivery system of the invention. U.S. Pat. No.
5,501,856 discloses controlled release pharmaceutical preparations
for intraocular implants to be applied to the interior of the eye
after a surgical operation for disorders in retina/vitreous body or
for glaucoma. U.S. Pat. No. 5,869,079 discloses combinations of
hydrophilic and hydrophobic entities in a biodegradable sustained
release implant, and describes a polylactic acid polyglycolic acid
(PLGA) copolymer implant comprising dexamethasone. As shown by in
vitro testing of the drug release kinetics, the 100-120 .mu.g 50/50
PLGA/dexamethasone implant disclosed did not show appreciable drug
release until the beginning of the fourth week, unless a release
enhancer, such as HPMC was added to the formulation. U.S. Pat. No.
5,824,072 discloses implants for introduction into a suprachoroidal
space or an avascular region of the eye, and describes a
methylcellulose (i.e. non-biodegradable) implant comprising
dexamethasone. WO 9513765 discloses implants comprising active
agents for introduction into a suprachoroidal or an avascular
region of an eye for therapeutic purposes. U.S. Pat. Nos. 4,997,652
and 5,164,188 disclose biodegradable ocular implants comprising
microencapsulated drugs, and describes implanting microcapsules
comprising hydrocortisone succinate into the posterior segment of
the eye. U.S. Pat. No. 5,164,188 discloses encapsulated agents for
introduction into the suprachoroid of the eye, and describes
placing microcapsules and plaques comprising hydrocortisone into
the pars plana. U.S. Pat. Nos. 5,443,505 and 5,766,242 disclose
implants comprising active agents for introduction into a
suprachoroidal space or an avascular region of the eye, and
describes placing microcapsules and plaques comprising
hydrocortisone into the pars plana. Zhou et al. disclose a
multiple-drug implant comprising 5-fluorouridine, triamcinolone,
and human recombinant tissue plasminogen activator for intraocular
management of proliferative vitreoretinopathy (PVR). Zhou, T, et
al. (1998). Development of a multiple-drug delivery implant for
intraocular management of proliferative vitreoretinopathy, Journal
of Controlled Release 55: 281-295. U.S. Pat. No. 6,369,116
discusses an implant with a release modifier inserted within a
scleral flap. EP 0 654256 discusses use of a scleral plug after
surgery on a vitreous body, for plugging an incision. U.S. Pat. No.
4,863,457 discusses the use of a bioerodible implant to prevent
failure of glaucoma filtration surgery by positioning the implant
either in the subconjunctival region between the conjunctival
membrane overlying it and the sclera beneath it or within the
sclera itself within a partial thickness sclera flap. EP 488 401
discusses intraocular implants, made of certain polylactic acids,
to be applied to the interior of the eye after a surgical operation
for disorders of the retina/vitreous body or for glaucoma. EP
430539 discusses use of a bioerodible implant which is inserted in
the suprachoroid.
[0151] The amount of drug delivery system administered will
typically depend upon the desired properties of the biodegradable
implant For example, the amount of drug delivery system can
influence the length of time in which the antiangiogenic
polypeptide component is released from the biodegradable implant
Additionally, the amount of drug delivery system administered will
typically depend upon the specific intended use (e.g., nature and
stage/progression of the disease or disorder).
[0152] Specifically, the drug delivery system can be formulated to
provide an implant that releases therapeutically effective amounts
of an antiangiogenic polypeptide for at least one week, two weeks,
one month, two months, three months, four months, five months, six
months, nine months, twelve months or more. Specifically, the drug
delivery system can be formulated for administration less than
about once per day. More specifically, the drug delivery system can
be formulated for administration less than about once per week,
less than about once per month, more than about once per year,
about once per week to about once per year, or about once per month
to about once per year.
[0153] In some embodiments, less than 5 ml, 4 ml, 3 ml, 2 ml, 1 ml,
0.1 ml, 0.01 ml, or 0.001 ml is administered. Specifically, the
drug delivery system administered can range from about 0.01 mL to
about 10.0 mL, about 0.05 mL to about 1.5 mL, about 0.1 mL to about
1.0 mL, or about 0.2 mL to about 0.8 mL.
[0154] The antiangiogenic polypeptide component can be present in
any effective, suitable and appropriate amount. For example,
polypeptide component can be present up to about 70 wt. % of the
drug delivery system, up to about 60 wt. % of the drug delivery
system, up to about 40 wt. % of the drug delivery system, up to
about 20 wt. % of the drug delivery system, 10 wt. % of the drug
delivery system, up to about 5 wt. % of the drug delivery system,
up to about 1 wt. % of the drug delivery system, or up to about 0.1
wt. % of the drug delivery system.
[0155] The drug delivery system will effectively deliver the
antiangiogenic polypeptide component to mammalian tissue at a
suitable, effective, safe, and appropriate dosage. For example, the
drug delivery system can effectively deliver the antiangiogenic
polypeptide component to mammalian tissue at a dosage of more than
about 0.001 picogram/kilogram/day, more than about 0.01
picogram/kilogram/day, more than about 0.1 picogram/kilogram/day,
or more than about 1 picogram/kilogram/day. Alternatively, the drug
delivery system can effectively deliver the antiangiogenic
polypeptide component to mammalian tissue at a dosage of up to
about 100 milligram/kilogram/day, up to about 50
milligram/kilogram/day, up to about 10 milligram/kilogram/day, or
up to about 1 milligram/kilogram/day.
[0156] More specifically, the drug delivery system can effectively
deliver the antiangiogenic polypeptide component to mammalian
tissue at a dosage of about 0.001 picogram/kilogram/day to about
100 milligram/kilogram/day; about 0.01 picogram/kilogram/day to
about 50 milligram/kilogram/day; about 0.1 picogram/kilogram/day to
about 10 milligram/kilogram/day; or about 1 picogram/kilogram/day
to about 1 milligram/kilogram/day.
[0157] The sustained-release intraocular drug delivery system can
further comprise analgesics, anesthetics, anti-infective agents, or
anti-steroidal agents. Suitable analgesics include, e.g.,
acetaminophen, phenylpropanolamine HCl, chlorpheniramine maleate,
hydrocodone bitartrate, acetaminophen elixir, diphenhydramine HCl,
pseudoephedrine HCl, dextromethorphan HBr, guaifenesin, doxylamine
succinate, pamabron, clonidine hydrochloride, tramadol
hydrochloride, carbamazepine, sodium hyaluronate, lidocaine, hylan,
Arnica Montana, radix (mountain arnica), Calendula officinalis
(marigold), Hamamelis (witch hazel), Millefolium (milfoil),
Belladonna (deadly nightshade), Aconitum napellus (monkshood),
Chamomilla (chamomile), Symphytum officinale (comfrey), Bellis
perennis (daisy), Echinacea angustifolia (narrow-leafed cone
flower), Hypericum perforatum (St. John's wort), Hepar sulphuris
calcareum (calcium sulfide), buprenorphine hydrochloride,
nalbuphine hydrochloride, pentazocine hydrochloride,
acetylsalicylic acid, salicylic acid, naloxone hydrochloride, oral
transmucosal fentanyl citrate, morphine sulfate, propoxyphene
napsylate, propoxyphene hydrochloride, meperidine hydrochloride,
hydromorphone hydrochloride, fentanyl transdermal system,
levorphanol tartrate, promethazine HCl, oxymorphone hydrochloride,
levomethadyl acetate hydrochloride, oxycodone HCl, oxycodone,
codeine phosphate, isometheptene mucate, dichloralphenazone,
butalbital, naproxen sodium, diclofenac sodium, misoprostol,
diclofenac potassium, celecoxib, sulindac, oxaprozin, salsalate,
diflunisal, naproxen, piroxicam, indomethacin, indomethacin sodium
trihydrate, etodolac, meloxicam, ibuprofen, fenoprofen calcium,
ketoprofen, mefenamic acid, nabumetone, tolmetin sodium, ketorolac
tromethamine, choline magnesium trisalicylate, and rofecoxib.
[0158] Suitable anesthetics include: propofol, halothane,
desflurane, midazolam HCl, epinephrine, levobupivacaine, etidocaine
hydrochloride, ropivacaine HCl, chloroprocaine HCl, bupivacaine
HCl, and lidocaine HCl.
[0159] Suitable anti-infective agents include, e.g., trimethoprim,
sulfamethoxazole, clarithromycin, ganciclovir sodium, ganciclovir,
daunorubicin citrate liposome, fluconazole, doxorubicin HCl
liposome, foscamet sodium, interferon alfa-2b, atovaquone,
rifabutun, trimetrexate glucoronate, itraconazole, ciclofovir,
azithromycin, delavirdine mesylate, efavirenz, nevirapine,
lamivudine/zidovudine, zalcitabine, didanosine, stavudine, abacavir
sulfate, amprenavir, indinavir sulfate, saquinavir, saquinavir
mesylate, ritonavir, nelfinavir, chloroquine hydrochloride,
metronidazole, metronidazole hydrochloride, iodoquinol,
albendazole, praziquantel, thiabendazole, ivermectin, mebendazole
sulfate, tobramycin sulfate, tobramycin, azetreonam, cefotetan
disodium, cefotetan, loracarbef, cefoxitin, meropenem, imipenemand
cilastatin, cefazolin, cefaclor, ceftibuten, ceftizoxime,
cefoperazone, cefuroxumeaxetil, cefprozil, ceftazidime, cefotaxime
sodium, cefadroxil monohydrate, cephalexin, cephalexin
hydrochloride, cefuroxime, cefazolin, cefamandole nafate, cefapime
hydrochloride, cefdinir, ceftriaxone sodium, cefixme, cefpodoxime
proxetil, dirithromycin, erythromycin, erythromycin ethylsuccinate,
erythromycin stearate, erythromycin, sulfisoxazole acetyl,
troleandomycin, azithromycin, clindamycin, clindamycin
hydrochloride, colistimethate sodium, quinupristin/dalfopristin,
vancomycin hydrochloride, amoxicillin,
amoxicillin/calvulanate/potassium, penicillin G benzathine,
penicillin G procaine, penicillin G potassium, carbenicillin
indanyl sodium, piperacillin sodium, ticarcillin disodium,
clavulanate potassium, ampicillin sodium/sulbactam sodium,
tazobactam sodium, tetracycline HCl, demeclocycline hydrochloride,
doxycycline hyclate, minocycline HCl, doxycycline monohydrate,
oxytetracycline HCl, hydrocortisone acetate, doxycycline calcium,
amphotericin B lipid, flucytosine, griseofulvin, terbinafine
hydrochloride, ketoconazole, chloroquine hydrochloride, chloroquine
phosphate, pyrimethamine, mefloquine hydrochloride, atovaquone and
proguanil hydrochloride, hydroxychloroquine sulfate, ethambutol
hydrochloride, aminosalicylic acid, rifapentine, rifampin,
isoniazid, pyrazinamide, ethionamide, interferon alfa-n3,
famciclovir, rimantadine hydrochloride, foscamet sodium, interferon
alfacon-1, ribavirin, zanamivir, amantadine hydrochloride,
palivizumab, oseltamivir phosphate, valacyclovir hydrochloride,
nelfinavir mesylate, stavudine, acyclovir, acyclovir sodium,
rifabutin, trimetrexate glucuronate, linezolid, moxifloxacin,
moxifloxacin hydrochloride, ciprofloxacin, ciprofloxacin
hydrochloride, ofloxacin, levofloxacin, lomefloxacin hydrochloride,
nalidixic acid, norfloxacin, enoxacin, gatifloxacin, trovafloxacin
mesylate, alatrofloxacin, sparfloxacin, aztreonam, nitrofurantoin
monohydrate/macrocrystals, cefepime hydrochloride, fosfomycin
tromethamine, neomycin sulfate-polymyxin B sulfate, imipenem,
cilastatin, methenamine, methenamine mandelate, phenyl salicylate,
atropine sulfate, hyoscyamine sulfate, benzoic acid,
oxytetracycline hydrochloride, sulfamethizole, phenazopyridine
hydrochloride, and sodium acid phosphate, monohydrate.
[0160] The steroidal anti-inflammatory agents that may be used in
the ocular implants include, but are not limited to,
21-acetoxypregnenolone, alclometasone, algestone, amcinonide,
beclomethasone, betamethasone, budesonide, chloroprednisone,
clobetasol, clobetasone, clocortolone, cloprednol, corticosterone,
cortisone, cortivazol, deflazacort, desonide, desoximetasone,
dexamethasone, diflorasone, diflucortolone, difluprednate,
enoxolone, fluazacort, flucloronide, flumethasone, flunisolide,
fluocinolone acetonide, fluocinonide, fluocortin butyl,
fluocortolone, fluorometholone, fluperolone acetate, fluprednidene
acetate, fluprednisolone, flurandrenolide, fluticasone propionate,
formocortal, halcinonide, halobetasol propionate, halometasone,
halopredone acetate, hydrocortamate, hydrocortisone, loteprednol
etabonate, mazipredone, medrysone, meprednisone,
methylprednisolone, mometasone furoate, paramethasone,
prednicarbate, prednisolone, prednisolone 25-diethylamino-acetate,
prednisolone sodium phosphate, prednisone, prednival, prednylidene,
rimexolone, tixocortol, triamcinolone, triamcinolone acetonide,
triamcinolone benetonide, triamcinolone hexacetonide, and any of
their derivatives.
Exemplary Uses
[0161] The small size and stable structure of the disclosed
polypeptides can be particularly valuable with respect to
manufacturing of the drug, rapid clearance from the body for
certain applications where rapid clearance is desired or
formulation into novel delivery systems that are suitable or
improved using a molecule with such characteristics.
[0162] On the basis of their efficacy as inhibitors of VEGF
biological activity, the polypeptides of the invention are
effective against a number of conditions associated with
inappropriate angiogenesis, including but not limited to autoimmune
disorders (e.g., rheumatoid arthritis, inflammatory bowel disease
or psoriasis); cardiac disorders (e.g., atherosclerosis or blood
vessel restenosis); retinopathies (e.g., proliferative
retinopathies generally, diabetic retinopathy, age-related macular
degeneration or neovascular glaucoma), renal disease (e.g.,
diabetic nephropathy, malignant nephrosclerosis, thrombotic
microangiopathy syndromes; transplant rejection; inflammatory renal
disease; glomerulonephritis; mesangioproliferative
glomerulonephritis; haemolytic-uraemic syndrome; and hypertensive
nephrosclerosis); hemangioblastoma; hemangiomas; thyroid
hyperplasias; tissue transplantations; chronic inflammation;
Meigs's syndrome; pericardial effusion; pleural effusion;
autoimmune diseases; diabetes; endometriosis; chronic asthma;
undesirable fibrosis (particularly hepatic fibrosis) and cancer, as
well as complications arising from cancer, such as pleural effusion
and ascites. Preferably, the VEGFR-binding polypeptides of the
invention can be used for the treatment of prevention of
hyperproliferative diseases or cancer and the metastatic spread of
cancers. Non-limiting examples of cancers include bladder, blood,
bone, brain, breast, cartilage, colon kidney, liver, lung, lymph
node, nervous tissue, ovary, pancreatic, prostate, skeletal muscle,
skin, spinal cord, spleen, stomach, testes, thymus, thyroid,
trachea, urogenital tract, ureter, urethra, uterus, or vaginal
cancer. Additional treatable conditions can be found in U.S. Pat.
No. 6,524,583, herein incorporated by reference. Other references
describing uses for VEGFR-2 binding polypeptides include: McLeod D
S et al., Invest Opthalmol V is Sci. 2002 February; 43(2):474-82;
Watanabe et al. Exp Dermatol. 2004 Nov.; 13(11):671-81; Yoshiji H
et al., Gut. 2003 September; 52(9):1347-54; Verheul et al.,
Oncologist. 2000; 5 Suppl 1:45-50; Boldicke et al., Stem Cells.
2001; 19(1):24-36.
[0163] As described herein, angiogenesis-associated diseases
include, but are not limited to, angiogenesis-dependent cancer,
including, for example, solid tumors, blood born tumors such as
leukemias, and tumor metastases; benign tumors, for example
hemangiomas, acoustic neuromas, neurofibromas, trachomas, and
pyogenic granulomas; inflammatory disorders such as immune and
non-immune inflammation; chronic articular rheumatism and
psoriasis; ocular angiogenic diseases, for example, diabetic
retinopathy, retinopathy of prematurity, macular degeneration,
corneal graft rejection, neovascular glaucoma, retrolental
fibroplasia, rubeosis; Osler-Webber Syndrome; myocardial
angiogenesis; plaque neovascularization; telangiectasia;
hemophiliac joints; angiofibroma; and wound granulation and wound
healing; telangiectasia psoriasis scleroderma, pyogenic granuloma,
cororany collaterals, ischemic limb angiogenesis, corneal diseases,
rubeosis, arthritis, diabetic neovascularization, fractures,
vasculogenesis, hematopoiesis.
[0164] In particular, the sustained-release intraocular drug
delivery system is useful for the treatment of retinopathies, such
as retinal vein occlusion, diabetic macular edema, diabetic
retinopathy, retinopathy of prematurity, macular degeneration,
age-related macular degeneration, corneal graft rejection,
neovascular glaucoma, retrolental fibroplasia, and rubeosis.
[0165] In one embodiment, the drug delivery system administers a
therapeutic component to ameliorate inflammation, and thus to
control, reduce or prevent an inflammatory response or ameliorate
the effects of an inflammatory response. In one embodiment, the
therapeutic component is used to enhance reabsorption of
inflammatory exudates. Decreasing the level of exudates in the eye
reduces the inflammatory process and the ensuing hyperpermeable
state that occurs with allergies, infection, responses to ocular
photodynamic therapy (PDT) and laser treatments, after ocular
surgery or trauma, etc.
[0166] In one embodiment, the therapeutic component is administered
to ameliorate the scarring and adhesions that are a part of the
inflammatory process. Adhesions are bands of scar tissue that bind
two internal body surfaces. They are an inflammatory response to
tissue damage, and occur as a normal part of any healing process.
As one example, adhesions frequently occur during the post-surgical
healing process during which tissues have experienced mechanical
trauma. However, adverse effects can occur when internal surfaces
bind, and adhesions may persist even after the original trauma has
healed. Surgery to repair adhesions itself results in recurrent or
additional adhesions. The presence of adhesions may also complicate
surgical procedures, for example, ocular conjunctival adhesions may
complicate subsequent glaucoma surgery.
[0167] Adhesions can occur following any type of trauma or surgery,
including but not limited to ocular surgery. Examples of ocular
surgery that may result in adhesions include glaucoma filtration
operations (i.e., iridencleisis and trephination, pressure control
valves), extraocular muscle surgery, diathermy or scleral buckling
surgery for retinal detachment, and vitreous surgery. Examples of
ocular trauma include penetrating ocular injuries, intraocular
foreign body, procedures such as PDT, scatter laser threshold
coagulation, refractive surgery, and blunt trauma.
[0168] In one embodiment, the therapeutic component ameliorates
disorders with both a vascular proliferative component and a
scarring component. As one example, the invention may be used in
patients with the ocular disease pterygia. In these patients,
fibrovascular proliferation results in scarring of the conjunctiva.
An elevated, superficial, external ocular mass, termed a pterygium,
forms and extends onto the corneal surface. Patients may experience
symptoms of inflammation (e.g., redness, swelling, itching,
irritation) and blurred vision. The mass itself may become
inflamed, resulting in redness and ocular irritation. Left
untreated, pterygia can distort the corneal topography, obscure the
optical center of the cornea, and result in altered vision.
[0169] The process whereby scar tissue forms (scarring) can occur
without new blood vessels being formed (neovascularization).
However, the neovascularization process always results in scarring
because of the cell proliferation that occurs with the formation of
new vessels also results in the proliferation of fibroblasts, glial
cells, etc. that result in scar tissue formation. The inventive
method may be used to ameliorate the scarring process.
[0170] In one embodiment, the therapeutic component is administered
to ameliorate inflammation of uveal tissues (uveitis, an
inflammation of tissues in the middle layer of the eye, mainly the
iris (iritis) and the ciliary body). Ocular inflammation may be
associated with underlying systemic disease or autoimmunity, or may
occur as a direct result of ocular trauma or infectious agents
(bacterial, viral, fungal, etc.). Inflammatory reactions in
adjacent tissues, e.g., keratitis, can induce a secondary uveitis.
There are both acute and chronic forms of uveitis. The chronic form
is frequently associated with many systemic disorders and most
likely occurs due to immunopathological mechanisms.
[0171] Uveitis presents with ocular pain, photophobia and
hyperlacrimation, with decreased visual acuity ranging from mild
blur to significant vision loss. Hallmark signs of anterior uveitis
are cells and flare in the anterior chamber. If the anterior
chamber reaction is significant, small gray to brown endothelial
deposits known as keratic precipitates may arise, leading to
endothelial cell dysfunction and corneal edema. There may be
adhesions to the lens capsule (posterior synechia) or the
peripheral cornea (anterior synechia). Granulomatous nodules may
appear on the surface of the iris stroma. Intraocular pressure is
initially reduced due to secretory hypotony of the ciliary body
but, as the reaction persists, inflammatory by-products may
accumulate in the trabeculum. If this debris builds significantly,
and if the ciliary body resumes its normal secretory output, the
pressure may rise sharply, resulting in a secondary uveitic
glaucoma.
[0172] A VEGFR-2 binding polypeptide can be administered alone or
in combination with one or more additional therapies such as
chemotherapy radiotherapy, immunotherapy, surgical intervention, or
any combination of these. Long-term therapy is equally possible as
is adjuvant therapy in the context of other treatment strategies,
as described above.
[0173] In certain embodiments of such methods, one or more
polypeptide therapeutic agents can be administered, together
(simultaneously) or at different times (sequentially). In addition,
polypeptide therapeutic agents can be administered with another
type of compounds for treating cancer or for inhibiting
angiogenesis.
[0174] In certain embodiments, the subject therapeutic agents of
the invention can be used alone. Alternatively, the subject agents
may be used in combination with conventional anti-cancer
therapeutic approaches directed to treatment or prevention of
proliferative disorders (e.g., tumor). For example, such methods
can be used in prophylactic cancer prevention, prevention of cancer
recurrence and metastases after surgery, and as an adjuvant of
other conventional cancer therapy. The present invention recognizes
that the effectiveness of conventional cancer therapies (e.g.,
chemotherapy, radiation therapy, phototherapy, immunotherapy, and
surgery) can be enhanced through the use of a subject polypeptide
therapeutic agent.
[0175] A wide array of conventional compounds have been shown to
have anti-neoplastic activities. These compounds have been used as
pharmaceutical agents in chemotherapy to shrink solid tumors,
prevent metastases and further growth, or decrease the number of
malignant cells in leukemic or bone marrow malignancies. Although
chemotherapy has been effective in treating various types of
malignancies, many anti-neoplastic compounds induce undesirable
side effects. It has been shown that when two or more different
treatments are combined, the treatments may work synergistically
and allow reduction of dosage of each of the treatments, thereby
reducing the detrimental side effects exerted by each compound at
higher dosages. In other instances, malignancies that are
refractory to a treatment may respond to a combination therapy of
two or more different treatments.
[0176] When the drug delivery system of the present invention is
administered in combination with a conventional anti-neoplastic
agent, either concomitantly or sequentially, such drug delivery
system may be found to enhance the therapeutic effect of the
anti-neoplastic agent or overcome cellular resistance to such
anti-neoplastic agent. This allows decrease of dosage of an
anti-neoplastic agent, thereby reducing the undesirable side
effects, or restores the effectiveness of an anti-neoplastic agent
in resistant cells.
[0177] The therapeutic agents that can be combined with the
sustained-release drug delivery system of the invention include
diverse agents used in oncology practice (Reference: Cancer,
Principles & Practice of Oncology, DeVita, V. T., Hellman, S.,
Rosenberg, S. A., 6th edition, Lippincott-Raven, Philadelphia,
2001), such as, merely to illustrate: abarelix, altretamine,
aminoglutethimide, amsacrine, anastrozole, antide, asparaginase,
AZD2171 (Recentin.TM.), Bacillus Calmette-Guerin/BCG (TheraCys.TM.,
TICE.TM.), bevacizumab (see U.S. Pat. No. 6,054,297; Avastin.TM.),
bicalutamide, bleomycin, bortezomib (Velcade.TM.), buserelin,
busulfan, campothecin, capecitabine, carboplatin, carmustine,
cetuximab (Erbitux.TM.), chlorambucil, cisplatin, cladribine,
clodronate, colchicine, cyclophosphamide, cyproterone, cytarabine,
dacarbazine, dactinomycin, dasatinib ((see U.S. Pat. No. 6,596,746
Sprycel.TM.), daunorubicin, dienestrol, diethylstilbestrol,
dexamethasone, docetaxel (Taxotere.TM.), doxorubicin, Abx-EGF,
epothilones, epirubicin, erlonitib (Tarceva.TM.), estradiol,
estramustine, etoposide, exemestane, 5-fluorouracil, filgrastim,
fludarabine, fludrocortisone, fluorouracil, fluoxymesterone,
flutamide, fulvestrant, gefitinib (Iressa.TM.), gemcitabine (see
U.S. Pat. No. 4,808,614; Gemzar.TM.), genistein, goserelin,
hydroxyurea, idarubicin, ifosfamide, imatinib mesylate (see U.S.
Pat. No. 5,521,184; Gleevac.TM.), interferon, irinotecan,
ibritumomab (Zevalin.TM.), ironotecan, ixabepilone (BMS-247550),
lapatinib (see U.S. Pat. No. 6,391,874; Tykreb.TM.), letrozole,
leucovorin, leuprolide, levamisole, lomustine, mechlorethamine,
medroxyprogesterone, megestrol, melphalan, mercaptopurine, mesna,
methotrexate, mitomycin, mitotane, mitoxantrone, motesanib
diphosphate (AMG 706) nilutamide, nocodazole, octreotide,
oxaliplatin, paclitaxel (Taxol.TM.), pamidronate, pentostatin,
plicamycin, porfimer, procarbazine, raltitrexed, rapamycin,
rituximab (Rituxan.TM.), sorafenib (Nexavar.TM./Bayer BAY43-9006),
streptozocin, suramin, sunitinib malate (see U.S. Pat. No.
6,573,293; Sutent.TM.), tamoxifen, temsirolimus (see U.S. Pat. No.
5,362,718; CCl-779), temozolomide (see U.S. Pat. No. 5,260,291;
Temodar.TM.), teniposide, testosterone, thioguanine, thiotepa,
titanocene dichloride, topotecan, toremifene, tositumomab
(Bexxar.TM.), trastuzumab (U.S. Pat. No. 5,821,337; Herceptin.TM.),
tretinoin, VEGF Trap (aflibercept; preparation described in U.S.
Pat. No. 5,844,099), vinblastine, vincristine, vindesine, and
vinorelbine, zoledronate.
[0178] Certain chemotherapeutic anti-tumor compounds may be
categorized by their mechanism of action into, for example,
following groups: anti-metabolites/anti-cancer agents, such as
pyrimidine analogs (5-fluorouracil, floxuridine, capecitabine,
gemcitabine and cytarabine) and purine analogs, folate antagonists
and related inhibitors (mercaptopurine, thioguanine, pentostatin
and 2-chlorodeoxyadenosine (cladribine));
antiproliferative/antimitotic agents including natural products
such as vinca alkaloids (vinblastine, vincristine, and
vinorelbine), microtubule disruptors such as taxane (paclitaxel,
docetaxel), vincristin, vinblastin, nocodazole, epothilones and
navelbine, epidipodophyllotoxins (etoposide, teniposide), DNA
damaging agents (actinomycin, amsacrine, anthracyclines, bleomycin,
busulfan, camptothecin, carboplatin, chlorambucil, cisplatin,
cyclophosphamide, cytoxan, dactinomycin, daunorubicin, doxorubicin,
epirubicin, hexamethylmelamineoxaliplatin, iphosphamide, melphalan,
merchlorehtamine, mitomycin, mitoxantrone, nitrosourea, plicamycin,
procarbazine, taxol, taxotere, teniposide,
triethylenethiophosphoramide and etoposide (VP 16)); antibiotics
such as dactinomycin (actinomycin D), daunorubicin, doxorubicin
(adriamycin), idarubicin, anthracyclines, mitoxantrone, bleomycins,
plicamycin (mithramycin) and mitomycin; enzymes (L-asparaginase
which systemically metabolizes L-asparagine and deprives cells
which do not have the capacity to synthesize their own asparagine);
antiplatelet agents; antiproliferative/antimitotic alkylating
agents such as nitrogen mustards (mechlorethamine, cyclophosphamide
and analogs, melphalan, chlorambucil), ethylenimines and
methylmelamines (hexamethylmelamine and thiotepa), alkyl
sulfonates-busulfan, nitrosoureas (carmustine (BCNU) and analogs,
streptozocin), trazenes--dacarbazinine (DTIC);
antiproliferative/antimitotic antimetabolites such as folic acid
analogs (methotrexate); platinum coordination complexes (cisplatin,
carboplatin), procarbazine, hydroxyurea, mitotane,
aminoglutethimide; hormones, hormone analogs (estrogen, tamoxifen,
goserelin, bicalutamide, nilutamide) and aromatase inhibitors
(letrozole, anastrozole); anticoagulants (heparin, synthetic
heparin salts and other inhibitors of thrombin); fibrinolytic
agents (such as tissue plasminogen activator, streptokinase and
urokinase), aspirin, dipyridamole, ticlopidine, clopidogrel,
abciximab; antimigratory agents; antisecretory agents (breveldin);
immunosuppressives (cyclosporine, tacrolimus (FK-506), sirolimus
(rapamycin), azathioprine, mycophenolate mofetil); anti-angiogenic
compounds (TNP-470, genistein) and growth factor inhibitors (e.g.,
VEGF inhibitors, fibroblast growth factor (FGF) inhibitors);
angiotensin receptor blocker; nitric oxide donors; anti-sense
oligonucleotides; antibodies (trastuzumab); cell cycle inhibitors
and differentiation inducers (tretinoin); mTOR inhibitors,
topoisomerase inhibitors (doxorubicin (adriamycin), amsacrine,
camptothecin, daunorubicin, dactinomycin, eniposide, epirubicin,
etoposide, idarubicin and mitoxantrone, topotecan, irinotecan),
corticosteroids (cortisone, dexamethasone, hydrocortisone,
methylpednisolone, prednisone, and prenisolone); growth factor
signal transduction kinase inhibitors; mitochondrial dysfunction
inducers and caspase activators; and chromatin disruptors.
[0179] In certain embodiments, pharmaceutical compounds that may be
used for combinatory anti-angiogenesis therapy include: (1)
inhibitors of release of "angiogenic molecules," such as bFGF
(basic fibroblast growth factor); (2) neutralizers of angiogenic
molecules, such as an anti-.beta.bFGF antibodies; and (3)
inhibitors of endothelial cell response to angiogenic stimuli,
including collagenase inhibitor, basement membrane turnover
inhibitors, angiostatic steroids, fungal-derived angiogenesis
inhibitors, platelet factor 4, thrombospondin, arthritis drugs such
as D-penicillamine and gold thiomalate, vitamin D.sub.3 analogs,
alpha-interferon, and the like. For additional proposed inhibitors
of angiogenesis, see Blood et al., Bioch. Biophys. Acta.,
1032:89-118 (1990), Moses et al., Science, 248:1408-1410 (1990),
Ingber et al., Lab. Invest., 59:44-51 (1988), and U.S. Pat. Nos.
5,092,885, 5,112,946, 5,192,744, 5,202,352, and 6573256. In
addition, there are a wide variety of compounds that can be used to
inhibit angiogenesis, for example, endostatin protein or
derivatives, lysine binding fragments of angiostatin, melanin or
melanin-promoting compounds, plasminogen fragments (e.g., Kringles
1-3 of plasminogen), tropoin subunits, antagonists of vitronectin
.alpha..sub.v.beta..sub.3, peptides derived from Saposin B,
antibiotics or analogs (e.g., tetracycline, or neomycin),
dienogest-containing compositions, compounds comprising a MetAP-2
inhibitory core coupled to a peptide, the compound EM-138, chalcone
and its analogs, and naaladase inhibitors. See, for example, U.S.
Pat. Nos. 6,395,718, 6,462,075, 6,465,431, 6,475,784, 6,482,802,
6,482,810, 6,500,431, 6,500,924, 6,518,298, 6,521,439, 6,525,019,
6,538,103, 6,544,758, 6,544,947, 6,548,477, 6,559,126, and
6,569,845.
[0180] Depending on the nature of the combinatory therapy,
administration of the drug delivery system of the invention may be
continued while the other therapy is being administered and/or
thereafter. Administration of the drug delivery system may be made
in a single dose, or in multiple doses. In some instances,
administration of the drug delivery system is commenced at least
several days prior to the conventional therapy, while in other
instances, administration is begun either immediately before or at
the time of the administration of the conventional therapy.
EXAMPLES
[0181] The following examples are for the purposes of illustrating
the invention, and should not be construed as limiting.
Example 1
Initial Identification of KDR Binding Molecules
[0182] A library of approximately 1013 RNA-protein fusion variants
was constructed based on the scaffold of the tenth type 3 domain of
human fibronectin with three randomized regions at positions 23-29,
52-55 and 77-86 (amino acid nos. are referenced to SEQ ID NO:5)
(three loop library; Xu et al, Chemistry & Biology 9:933-942,
2002). Similar libraries were constructed containing randomized
regions only at positions 23-29 and 77-86 (two loop library) or
only at positions 77-86 (one loop library). A mixture of these
three libraries was used for in vitro selection against the
extracellular domain of human VEGFR-2 (KDR, extracellular domain,
residues 1-764 fused to human IgG1 Fc). For the purposes of this
application, the amino acid positions of the loops will be defined
as residues 23-30 (BC Loop), 52-56 (DE Loop) and 77-87 (FG Loop).
The target binding population was analyzed by DNA sequencing after
six rounds of selection and was found to be diverse, with some
replicates present. Proteins encoded by fifteen independent clones
were screened for binding to KDR, (FIG. 1A) and the best binders
were subsequently analyzed for inhibition of target binding in the
presence of VEGF (FIG. 1B). Multiple clones were identified that
inhibited KDR-VEGF binding, suggesting that these clones bound KDR
at or near the natural ligand (VEGF) binding site. The ability of
two of the binding molecules (VR28 and VR12) to directly inhibit
VEGF-KDR interaction was evaluated in a BIAcore assay using
immobilized VEGF and a mobile phase containing KDR-Fc with or
without a selected binding protein. VR28 and, to a lesser extent,
VR12, but not a non-competing clone (VR17), inhibited KDR binding
to VEGF in a dose dependent manner (FIG. 1C). Finally, in addition
to binding to purified recombinant KDR, VR28 also appeared to bind
to KDR-expressing recombinant CHO cells, but not to control CHO
cells (FIG. 1D).
[0183] The sequence of the binding loops of the VR28 clone is shown
in the first row of Table 4. While VR28 was not the most abundant
clone in the sequenced binding population (one copy out of 28
sequenced clone), its binding affinity to KDR was the best among
the tested clones from this binding population, with a dissociation
constant of 11-13 nM determined in a radioactive equilibrium
binding assay (FIG. 3 and Table 5) and BIAcore assays (Table 7).
There were no changes from wild type .sup.10Fn3 in the remaining
scaffold portion of the molecule (following correction of an
incidental scaffold change at position 69 that had no effect on
binding). However, VR28 showed little inhibition of VEGF-KDR
signaling in a VEGF-dependent cell proliferation assay. Thus, while
the selection from the naive library yielded antibody mimics that
interfered with the interaction between VEGF and KDR in biochemical
binding studies, affinity improvements were useful for neutralizing
function in a biological signal transduction assay.
Example 2
Affinity Maturation of Clone VR28
[0184] A mutagenesis strategy focusing on altering sequences only
in the binding loops was employed. To initially test which loops
were more likely to result in improvement, loop-directed
hypermutagenic PCR was carried out to introduce up to 30% mutations
independently into each loop of VR28. After three rounds of
selection against KDR, multiple clones with improved binding to
KDR-Fc were observed. Sequence analysis of the selection pools
revealed that the majority of mutations were accumulated in the FG
loop while the BC and DE loops remained almost intact. This result
indicated that the FG loop was the most suitable target for further
modification.
[0185] Consequently, a new library of approximately 1012 variants
was constructed by altering the sequence of VR28 in the FG loop
using oligonucleotide mutagenesis. For each of the FG loop
positions (residues 77-86 [VAQNDHELIT (SEQ ID NO:198)] as well as
the following Proline [residue 87]), a 50:50 mixture of the
VR28-encoding DNA and NNS was introduced at each position. DNA
sequence analysis of a random sample of approximately 80 clones
revealed an average of six amino acid changes per clone as
expected. Lower KDR-Fc concentrations were utilized during
selection to favor clones with better affinities to the target. The
profile of target binding during the four rounds of selection is
shown in FIG. 2. After four rounds of selection the binding
population was subcloned and analyzed. Table 5 and FIG. 3A
summarize affinity measurements of individual binding clones. The
measured binding constants to KDR-Fc ranged from <0.4 to <1.8
nM, a 10-30 improvement over VR28 (11 nM).
[0186] Sequence analysis, some of which is shown in Table 4 (K
clones), revealed that while the binding population was diverse,
several consensus motifs could be identified among the clones. Most
noticeably, Pro87 and Leu84 were found in nearly all clones (as in
VR28), suggesting that these residues may be essential for the
structure of the binding site. A positively charged amino acid at
position 82 appears to be required since only H82K or H82R changes
were seen in the sequenced clones and an aliphatic amino acid was
predominant at position 78. D81 was often mutated to a G, resulting
in the loss of negative charge at this position and a gain in
flexibility. In addition, the overall mutation rate in the selected
population was comparable to the pool prior to selection, which
suggested that the FG loop is very open to changes.
[0187] Several residues in the N-terminus of the .sup.10Fn3 domain
of human fibronectin are located in close proximity to the FG loop,
as suggested by structural determinations (Main et al, Cell
71:671-678, 1992). The close proximity of the two regions could
potentially have a negative impact on target binding. Two
incidental mutations in the N-terminal region, L8P and L8Q,
resulted in better binding to KDR in a number of selected clones,
presumably due to a change of the location of the N-terminus
relative to the FG loop. To further test the impact of the
N-terminus, we created binding molecules for 23 different KDR
binders in which the N-terminal first eight residues before the
.beta.-sheet were deleted. We then compared target binding to the
non-deleted counterparts. On average, binding to KDR-Fc was about
3-fold better with the deletion, as shown in FIG. 3B.
Example 3
Selection of Binders with Dual Specificities to Human (KDR) and
Mouse (Flk-1) VEGFR-2
[0188] VR28 and most of the affinity matured variants (K clones)
failed to bind the mouse homolog of KDR, Flk1, as shown in FIG. 4.
However, since KDR and Flk1 share a high level of sequence identity
(85%, Claffey et al., J. Biol. Chem. 267:16317-16322 (1992), Shima
et al., J. Biol. Chem. 271:3877-3883 (1996)), it is conceivable to
isolate antibody mimics that can bind both KDR and Flk1. Such dual
binders were desirable because they would allow the same molecule
to be tested in functional studies in animal models and
subsequently in humans.
[0189] The population of clones following FG loop mutagenesis and
selection against KDR for four rounds was further selected against
Flk1 for an additional three rounds. As shown in FIG. 2 an increase
in binding to Flk1 was observed from Round 5 to Round 7, indicating
enrichment of Flk1 binders. Analysis of binding for multiple
individual clones revealed that in contrast to the clones selected
against KDR only (K clones), most clones derived from additional
selection against Flk1 (E clones) are able to interact with both
KDR and Flk1. The binding constants to both targets, as determined
using a radioactive equilibrium binding assay (Table 6 and FIG. 5)
and BIAcore (Table 7), indicate that individual clones were able to
bind both targets with high affinities.
[0190] For example, E19 has a Kd of 60 pM to KDR, and 340 pM to
Flk-1. These results demonstrate that a simple target switch
strategy in the selection process, presumably through selection
pressures exerted by both targets, has allowed the isolation of
molecules with dual binding specificities to both KDR and Flk-1
from a mutagenized population of VR28, a moderate KDR binder that
was not able to bind Flk-1. The selected fibronectin-based binding
proteins are highly specific to VEGFR-2 (KDR) as no substantial
binding to VEGFR1 was observed at high target concentration.
Sequence analysis revealed some motifs similar to those observed in
the KDR binder pool (Leu and Pro at residues 84 and 87
respectively; positively charged amino acid at residue 82,
predominantly Arg) and some that were not maintained (aliphatic at
position 78). In addition, the motif ERNGR (residues 78-82) was
present in almost all clones binding to Flk-1 (Table 4); this motif
was barely discernable in the KDR binding pool. R79 and R82 appear
to be particularly important for high affinity binding to Flk-1,
since binding to Flk-1, but not KDR, is greatly reduced when a
different residue is present at this position (FIG. 6A). To
determine the importance of each loop in binding to KDR and Flk-1,
the loops of clones E6 and E26 shown in Table 4, were substituted
one loop at a time by NNS randomized sequence. As shown in FIG. 6B,
after the substitution, the proteins are no longer able to bind
either KDR or Flk-1. These results indicate that each loop is
required for binding to the targets, suggesting a cooperative
participation of all three loops in interacting with the
targets.
[0191] An alternative mutagenesis strategy was independently
employed to produce clones capable of binding to both targets. The
clone 159Q(8)L (Table 4), the product of hypermutagenic PCR
affinity maturation of VR28 that binds KDR with high affinity (Kd=2
nM; Table 7) and Flk-1 with poor affinity (Kd>3000 nM), was
chosen as a starting point. The first six amino acids of the FG
loop were fully randomized (NNS), leaving the following five
residues (ELFTP) intact. After six rounds of selection against
Flk-1, the binding pool was re-randomized at the DE loop (positions
52-56) and the selection was performed for three additional rounds
against Flk-1 and one round against KDR. A number of high affinity
binding molecules to both KDR and Flk-1 were thus obtained (Tables
4 and FIG. 4). For example, clone M5FL, while retaining high
binding affinity to KDR (Kd=890 pM), can bind Flk-1 at a Kd of 2.1
nM, a 1000-fold improvement over the original clone. Interestingly,
the ERNGR motif, found in Flk-1 binding molecules selected from a
mutagenized population of VR28, was also present in multiple clones
derived from clone 159Q(8)L mutagenesis and selection, despite a
full randomization of this region of the FG loop. The isolation of
similar binding molecules from two independent libraries suggests
that the affinity maturation process is robust for isolating
optimal Flk-1 binding motifs located in the FG loop.
Example 4
Cell Surface Binding and Neutralization of VEGF Activity In
Vitro
[0192] The functionality of KDR and Flk-1 binding molecules in a
cell culture model system was evaluated with E. coli produced
binding molecules. Using a detection system consisting of anti-His6
tag murine antibody (the E. coli expressed proteins were expressed
with a His tag) and an anti-murine fluorescently labeled antibody
the binding molecules were shown to bind specifically to mammalian
cells expressing KDR or Flk-1 with low nanomolar EC50s, (FIG. 7 and
Table 8).
[0193] More importantly, using recombinant BA/F3 cells
(DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH)
expressing the extracellular KDR or Flk-1 domain linked to the
erythropoietin receptor signaling domain, these molecules inhibited
VEGF-stimulated cell proliferation in a dose dependent fashion,
with IC50 3-12 nM for KDR expressing cells, and 2-5 nM for Flk-1
expressing cells. The potency of inhibition appears to be similar
to control anti-KDR and anti-Flk-1 monoclonal antibodies, as shown
in FIG. 8 and Table 9.
[0194] A number of clones were further tested for VEGF-inhibition
of the growth of HUVEC cells (Human Umbilical Vein Endothelial
Cells). HUVEC cells are natural human cells that are closely
related to cells in the body that respond to VEGF. As shown in FIG.
9 and Table 10, the fibronectin-based binding proteins were also
active in inhibiting VEGF activity in this human-derived cell
system while the wild type fibronectin-based scaffold protein was
inactive.
Example 5
Thermal Stability and Reversible Refolding of M5FL Protein
[0195] The thermal stability of KDR-binder M5FL was established
using differential scanning calorimetry (DSC). Under standard PBS
buffer conditions (sodium phosphate pH 7.4, 150 mM NaCl), M5FL was
found to have a single non-reversible thermal melting transition at
56.degree. C. Subsequently, sodium acetate pH 4.5 was identified as
a favorable buffer for M5FL protein solubility. DSC experiments in
this buffer (100 mM) demonstrated that M5FL is more stable under
these conditions (Tm=67-77.degree. C.) and that the melting
transition is reversible (FIG. 10). Reversible thermal transitions
have been used to identify favorable conditions that support
long-term storage of protein therapeutics (Remmele et al,
Biochemistry 38:5241 (1999), so Na-acetate pH 4.5 has been
identified as an optimized buffer for storing the M5FL protein.
Example 6
In Vitro Binding and Cell-Based Activity of PEGylated M5FL
Protein
[0196] The M5FL protein was produced in an E. coli expression
system with a C-terminal extension to yield the following protein
sequence (C-terminal extension underlined with Cys100 shaded; a
significant percentage of protein is produced with the initial
methionine removed):
TABLE-US-00010 (SEQ ID NO:199)
MGVSDVPRDLEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEF
TVPLQPPLATISGLKPGVDYTITVYAVTKERNGRELFTPISINYRTEIDK PCQHHHHHH
[0197] The single sulfhydryl of the cysteine residue at position
100 was used to couple to PEG variants using standard maleimide
chemistry to yield two different PEGylated forms of M5FL. Both a
linear 20 kD PEG and a branched 40 kD PEG (Shearwater Corporation)
were conjugated to M5FL to produce M5FL-PEG20 and M5FL-PEG40,
respectively. The PEGylated protein forms were purified from
unreacted protein and PEG by cation exchange chromatography.
Covalent linkage of the two PEG forms of M5FL was verified by
SDS-PAGE (FIG. 11) and mass spectroscopy.
[0198] In vitro affinity measurements were made using surface
plasmon resonance (SPR) (BIAcore) with both the human and mouse
VEGF-receptor target proteins immobilized via amide chemistry on
the BIAcore chip. For both target proteins, both the 20 and 40 kD
PEGylated M5FL forms were found to have slower on-rates (ka)
relative to unmodified M5FL with little effect on off-rates (kd;
Table 11).
[0199] The functionality of the PEGylated M5FL preparations was
tested using the Ba/F3 system described in Example 4. FIG. 12 shows
a plot of A490 (representing the extent of cell proliferation) as a
function of concentration of each of the binders. The curves were
nearly identical, indicating there was little effect of PEGylation
on the biological activity of either of the PEGylated forms.
[0200] The k.sub.on, k.sub.off and K.sub.D were analyzed for a
subset of KDR-binding polypeptides and compared to the EC50 for the
BaF3 cell-based VEGF inhibition assay. Scatter plots showed that
the kon was well-correlated with the EC50, while k.sub.off was
poorly correlated. Greater than 90% of KDR-binding proteins with a
kon of 105s.sup.-1 or greater had an EC50 of 10 nM or less. K.sub.D
is a ratio of kon and koff, and, as expected, exhibits an
intermediate degree of correlation with EC50.
[0201] Many of the KDR-binding proteins, including CT-01, were
assessed for binding to VEGFR-1, VEGFR-2 and VEGFR-3. The proteins
showed a high degree of selectivity for VEGFR-2.
Example 6
Preparation of KDR Binding Protein CT-01 Blocks VEGFR-2 Signaling
in Human Endothelial Cells
[0202] Following the methodologies described in the preceding
Examples, additional .sup.10Fn3-based KDR binding proteins were
generated. As described for the development of the M5FL protein in
Example 5, above, proteins were tested for K.sub.D against human
KDR and mouse Flk-1 using the BIAcore binding assay and for IC50 in
a Ba/F3 assay. A protein termed CT-01 exhibited desirable
properties in each of these assays and was used in further
analysis.
[0203] The initial clone from which CT-01 was derived had a
sequence:
GEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLK
PGVDYTITVYAVTDGWNGRLLSIPISINYRT (SEQ ID NO:200). The FG loop
sequence is underlined.
[0204] Affinity maturation as described above produced a core form
of CT-01:
TABLE-US-00011 (SEQ ID NO:192)
EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTAT
ISGLKPGVDYTITVYAVTDGRNGRLLSIPISINYRT.
[0205] The CT-01 molecule above has a deletion of the first 8 amino
acids and may include additional amino acids at the N- or
C-termini. For example, an additional MG sequence may be placed at
the N-terminus. The M will usually be cleaved off, leaving a GEV .
. . sequence at the N-terminus. The re-addition of the normal 8
amino acids at the N-terminus also produces a KDR binding protein
with desirable properties. The N-terminal methionine is generally
cleaved off to yield a sequence:
TABLE-US-00012 (SEQ ID NO:193)
VSDVPRDLEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTV
PLQPPTATISGLKPGVDYTITVYAVTDGRNGRLLSIPISTNYRT.
[0206] For use in vivo, a form suitable for PEGylation may be
generated. For example, a C-terminal tail comprising a cysteine was
added and expressed, as shown below for a form lacking the eight
N-terminal amino acids.
GEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLK
PGVDYTITVYAVTDGRNGRLLSIPISINYRTEIDKPCQ (SEQ ID NO:194). The
PEGylated form of this molecule is used in the in vivo experiments
described below. A control form with a serine instead of a cysteine
was also used:
TABLE-US-00013 (SEQ ID NO:195)
GEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTA
TISGLKPGVDYTITVYAVTDGRNGRLLSIPISTNYRTEIDKPSQ.
[0207] The same C-terminal tails may also be added to CT-01 forms
having the N-terminal eight amino acids, such as is shown in SEQ ID
NO:193.
[0208] Additional variants with desirable KDR binding properties
were isolated. The following core sequence has a somewhat different
FG loop, and may be expressed with, for example, an N-terminal MG
sequence, an N-terminal sequence that restores the 8 deleted amino
acids, and/or a C-terminal tail to provide a cysteine for
PEGylation.
EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKP
GVDYTITVYAVTEGPNERSLFIPISINYRT (SEQ ID NO:196). Another such
variant has the core sequence:
TABLE-US-00014 (SEQ ID NO:197)
VSDVPRDLEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTV
PLQPPTATISGLKPGVDYTITVYAVTEGPNERSLFIPISINYRT.
[0209] A comparison of these variants shows a consensus sequence
for the FG loop of: D/E)GXNXRXXIP (SEQ ID NO:3). With greater
particularity, the consensus sequence may be expressed as
(D/E)G(R/P)N(G/E)R(S/L)(S/F)IP (SEQ ID NO:4).
Example 7
CT-01 Blocks VEGFR-2 Signaling in Human Endothelial Cells
[0210] As shown in FIG. 13, VEGF-A signaling through VEGFR-2 is
mediated by phosphorylation of the intracellular domain of VEGFR-2,
followed by activation of pathway involving phospholipase C gamma
(PLC.gamma.), Protein Kinase C(PKC), Raf-1, MEK1/2, ERK1/2, leading
to endothelial cell proliferation.
[0211] To assess whether KDR binders disclosed herein inhibited
activation of this signaling pathway, Human Microvascular
Endothelial Cells were treated with a VEGFR binding polypeptide
(e.g., CT-01) for 30 min and stimulated with VEGF-A for 5 min.
Total cell lysates were analyzed by SDS-PAGE and western analysis,
using antibodies specific to phospho-VEGFR-2, non-phospho-VEGFR-2,
phosphor-ERK1/2 and non-phospho-ERK1/2.
[0212] As shown in FIG. 13, 130 pM CT-01 inhibits formation of
phosphor-VEGFR-2 and also decreases the formation of the downstream
phosphorylated ERK1/2. Phosphorylated ERK1/2 is not entirely
eliminated, probably due to the fact that ERK1/2 receives signals
from a number of additional signaling pathways.
Example 8
Fibronectin-based KDR Binding Proteins Disrupt Signaling by VEGF-A
and VEGF-D
[0213] VEGFR-2 is a receptor for three VEGF species, VEGF-A, VEGF-C
and VEGF-D.
[0214] Experiments were conducted to evaluate the effects of
fibronectin-based KDR binding proteins on VEGF-A and VEGF-D
mediated signaling through KDR.
[0215] A Ba/F3 cell line dependent on Flk-1 mediated signaling was
generated. As shown in the left panel of FIG. 14, cell viability
could be maintained by treating the cells with VEGF-A or VEGF-D,
although significantly higher levels of VEGF-D were required.
[0216] As shown in the middle panel of FIG. 14, cells were
maintained in the presence of 15 ng/ml of VEGF-A and contacted with
the M5FL or CT-01 proteins disclosed herein, or with the DC-101
anti-Flk-1 antibody. Each reagent reversed the VEGF-A-mediated cell
viability, indicating that VEGF-A signaling through Flk-1 was
blocked.
[0217] As shown in the right panel of FIG. 14, cells were
maintained in the presence of 300 ng/ml of VEGF-D and contacted
with the M5FL or F10 proteins disclosed herein, or with an
anti-VEGF-A antibody. M5FL and F10 reversed the VEGF-D-mediated
cell viability, indicating that VEGF-D signaling through Flk-1 was
blocked. The anti-VEGF-A antibody had no effect, demonstrating the
specificity of the assay.
Example 9
Pharmacokinetics
[0218] Pharmacokinetic Studies: Native CT-01 or a pegylated form
(40 kDa PEG, CT-01PEG40) were iodinated with .sup.125I. 10-20 mCi
of iodinated proteins were injected into adult male rats either
i.v. or i.p. and iodinated proteins levels were determined at the
indicated times. For tissue distribution studies, rats were
sacrificed at 15 min, 2 hr and 6 hr and radioactivity levels
determined. See FIGS. 15 and 16. Unmodified CT-01 is a 12 kDa
protein that is rapidly cleared from the blood. The
area-under-curve value (AUC) value is 14.6 hr*mg/mL with a
clearance of 69.9 mL/hr/kg, a maximum serum concentration of 9.1
mg/ml. The initial half-life (a) is 0.3 hours and the second phase
half-life (.beta.) is 13.5 hours. By comparison, i.v. PEGylated
CT-01 has greatly increased presence in the blood, mostly because
of a dramatic decrease in the initial phase of clearance. The AUC
is increased greater than 10 fold to 193, the clearance rate is
decreased by greater than 10 fold to 5.2, the Cmax is 12.9 mg/mL.
The .alpha. half-life is increased to 1 hour, and the .beta. is
increased to 16.2 hours. These pharmacokinetics in rats are
equivalent to a twice-weekly dosing regimen in humans, a rate of
dosing that is well within acceptable ranges.
[0219] Intraperitoneal (i.p.) administration of PEGylated CT-01 had
reservoir-like pharmacokinetics. There was no initial spike in the
blood concentration of CT-01. Instead, the amount of CT-01 built up
more slowly and decreased slowly. Such pharmacokinetics may be
desirable where there is concern about side effects from the
initial spike in CT-01 concentration upon intravenous
administration. It is likely that other .sup.10FN3-based agents
would exhibit similar behavior in i.p. administration.
[0220] Accordingly, this may be a generalizable mode for achieving
a time-delayed dosing effect with .sup.10FN3-based agents.
[0221] As shown in FIG. 16, the liver is the primary route for
secretion of the PEGylated form of CT-01. No long term accumulation
of CT-01 was detected.
[0222] Similar results were obtained using a CT-01 conjugated to a
20 kDa PEG moiety.
Example 10
In Vivo Efficacy of CT-01
[0223] The Miles assay, as outlined in FIG. 17, is used to evaluate
Dose, Schedule and Administration parameters for the tumor efficacy
studies. Balb/c female mice were injected i.p. with buffer or
CT-01PEG40 at 1, 5 and 20 mg/kg 4 hr prior to VEGF challenge.
Intradermal focal administration of VEGF-A into the back skin
induces vessel leakage of Evans blue dye (FIGS. 17 and 18).
[0224] Mice treated with a KDR binding agent showed a statistically
significant decrease in the level of VEGF-mediated vessel leakage.
Both 5 mg/kg and 20 mg/kg dosages with CT-01 showed significant
results. Therefore, a 5 mg/kg dosage was selected for mouse tumor
model studies.
Example 11
CT-01 Inhibits Tumor Growth
B 16-F10 Murine Melanoma Tumor Assay:
[0225] 2.times.10.sup.6 B16-F10 murine melanoma tumor cells were
implanted subcutaneously into C57/BL male mice at Day 1. At day 6 a
palpable mass was detected. On day 8 when tumors were of measurable
size, daily i.p. injections of either Vehicle control, 5, 15, or 40
mg/kg CT-01PEG40 were started. The lowest dose 5 mg/kg decreased
tumor growth.
[0226] At day 18, mice treated with 15 and 40 mg/kg showed 50% and
66% reduction in tumor growth. See FIG. 19.
U87 Human Glioblastoma Assay:
[0227] 5.times.10.sup.6 U87 human glioblastoma tumor cells were
implanted subcutaneously into nude male mice. When tumor volume
reached approximately 50 mm3 treatment started (day 0). Vehicle
control, 3, 10, or 30 mg/kg CT-01PEG40 were injected i.v. every
other day (EOD). The anti-Flk-1 antibody DC101 was injected at 40
mg/kg twice a week as published for its optimal dose schedule. The
lowest dose 3 mg/kg decreased tumor growth. At day 12, mice treated
with 10 and 30 mg/kg showed 50% reduction in tumor growth. See FIG.
20. Effectiveness is comparable to that of the anti-Flk-1
antibody.
[0228] The following materials and methods were used for the
experiments described in Examples 1-11.
Recombinant Proteins:
[0229] Recombinant human VEGF.sub.165, murine VEGF.sub.164, human
neurotrophin-4 (NT4), human and mouse vascular endothelial growth
factor receptor-2 Fc chimeras (KDR-Fc and Flk-1-Fc) were purchased
from R&D systems (Minneapolis, Minn.). Biotinylation of the
target proteins was carried out in 1.times.PBS at 4.degree. C. for
2 hours in the presence of EZ-Lik.TM. Sulfo-NHS-LC-LC-Biotin
(Pierce, Ill.). Excess of EZ-Link.TM. Sulfo-NHS-LC-LC-Biotin was
removed by dialysis against 1.times.PBS. The level of biotinylation
was determined by mass spectroscopy and target protein
concentrations were determined using Coomassie Protein Plus Assay
(Pierce, Ill.).
Primers:
[0230] The following oligonucleotides were prepared by chemical
synthesis for eventual use in library construction and mutagenesis
of selected clones.
TABLE-US-00015 T7 TMV Fn: 5' GCG TAA TAC GAC TCA CTA TAG GGA CAA
TTA CTA TTT ACA ATT ACA ATG GTT TCT GAT GTT CCG AGG 3' (SEQ ID
NO:201) T7 TMV N-terminus deletion: 5' GCG TAA TAC GAC TCA CTA TAG
GGA CAA TTA CTA TTT ACA ATT ACA ATG GAA GTT GTT GCT GCG ACC CCC ACC
AGC CTA 3' (SEQ ID NO:202) MK165-4 A20: 5' TTT TTT TTT TTT TTT TTT
TTA AAT AGC GGA TGC CTT GTC GTC GTC GTC CTT GTA GTC 3' (SEQ ID
NO:203) N-terminus forward: 5' ATG GTT TCT GAT GTT CCG AGG GAC CTG
GAA GTT GTT GCT GCG ACC CCC ACC AGC CTA CTG ATC AGC TGG 3' (SEQ ID
NO:204) BCDE reverse: 5' AGG CAC AGT GAA CTC CTG GAC AGG GCT ATT
TCC TCC TGT TTC TCC GTA AGT GAT CCT GTA ATA TCT 3' (SEQ ID NO:205)
BCDE forward: 5' AGA TAT TAC AGG ATC ACT TAC GGA GAA ACA GGA GGA
AAT AGC CCT GTC CAG GAG TTC ACT GTG CCT 3' (SEQ ID NO:206) DEFG
reverse: 5' AGT GAC AGC ATA CAC AGT GAT GGT ATA ATC AAC TCC AGG TTT
AAG GCC GCT GAT GGT AGC TGT 3' (SEQ ID NO:207) DEFG forward: 5' ACA
GCT ACC ATC AGC GGC CTT AAA CCT GGA GTT GAT TAT ACC ATC ACT GTG TAT
GCT GTC ACT 3' (SEQ ID NO:208) C-terminus polyA: 5' TTT TTT TTT TTT
TTT TTT TAA ATA GCG GAT GCC TTG TCG TCG TCG TCC TTG TAG TCT GTT CGG
TAA TTA ATG GAA AT 3' (SEQ ID NO:209) Hu3'FLAGSTOP: 5' TTT TAA ATA
GCG GAT GCC TTG TCG TCG TCG TCC TTG TAG TCT GTT CGG TAA TTA ATG G
3' (SEQ ID NO:210) R28FG-50: 5' GTG TAT GCT GTC ACT 123 145 463 665
165 465 163 425 625 645 447 ATT TCC ATT AAT TAC 3' , (SEQ ID
NO:211), where 1 = 62.5%G + 12.5%A + 12.5% T + 12.5%C; 2 = 2.5%G +
12.5%A + 62.5%T + 12.5%C; 3 = 75%G + 25%C; 4 = 12.5%G + 12.5%A +
12.5%T + 62.5%C; 5 = 25%G + 75%C; 6 = 12.5%G + 62.5%A + 12.5%T +
12.5%C; 7: 25%G + 50%A + 25%C F1U2: 5' TAA TAC GAC TCA CTA TAG GGA
CAA TTA CTA TTT ACA ATT CTA TCA ATA CAA TGG TGT CTG ATG TG CCG 3'
(SEQ ID NO:212) F2: 5' CCA GGA GAT CAG CAG GGA GGT CGG GGT GGC AGC
CAC CAC TTC CAG GTC GCG CGG CAC ATC AGA CAC CAT TGT 3' (SEQ ID
NO:213) F3159: 5' ACC TCC CTG CTG ATC TCC TGG CGC CAT CCG CAT TTT
CCG ACC CGC TAT TAC CGC ATC ACT TAC G 3' (SEQ ID NO:214) F4: 5' CAC
AGT GAA CTC CTG GAC CGG GCT ATT GCC TCC TGT TTC GCC GTA AGT GAT GCG
GTA ATA GCG 3' (SEQ ID NO:215) F5159: 5' CGG TCC AGG AGT TCA CTC
TGC CGC TGC AGC CGC CGG CGG CTA CCA TCA GCG GCC TTA AAC C 3' (SEQ
ID NO:216) F5-X5: 5' CG GTC CAG GAG TTC ACT GTG CCG NNS NNS NNS NNS
NNS GCT ACC ATC AGC GGC CTT AAA CC 3' (SEQ ID NO:217) F6: 5' AGT
GAC AGC ATA CAC AGT GAT GGT ATA ATC AAC GCC AGG TTT AAG GCC GCT GAT
GGT AG 3' (SEQ ID NO:218) F7X6159: 5' ACC ATC ACT GTG TAT GCT GTC
ACT NNS NNS NNS NNS NNS NNS GAA CTG TTT ACC CCA ATT TCC ATC AAC TAC
CGC ACA GAC TAC AAG 3' (SEQ ID NO:219) F8: 5' AAA TAG CGG ATG CGC
GTT TGT TCT GAT CTT CCT TAT TTA TGT GAT GAT GGT GGT GAT GCT TGT CGT
CGT CGT CCT TGT AGT CTG TGC GGT AGT TGA T 3' (SEQ ID NO:220)
G2asaiA20: 5' TTT TTT TTT TTT TTT TTT TTA AAT AGC GGA TGC GCG TTT
GTT CTG ATC TTC 3' (SEQ ID NO:221) C2RT: 5' GCG CGT TTG TTC TGA TCT
TCC 3' (SEQ ID NO:222) hf01 BC reverse: 5' TGCC TCC TGT TTC GCC GTA
AGT GAT GCG GTA ATA GCG SNN SNN SNN SNN SNN SNN SNN CCA GCT GAT CAG
CAG 3' (SEQ ID NO:223) hf01 DE reverse: 5' GAT GGT AGC TGT SNN SNN
SNN SNN AGG CAC AGT GAA CTC CTG GAC AGG GCT ATT GCC TCC TGT TTC GCC
3' (SEQ ID NO:224) hf01 FG reverse: 5' GT GCG GTA ATT AAT GGA AAT
TGG SNN SNN SNN SNN SNN SNN SNN SNN SNN SNN AGT GAC AGC ATA CAC 3'
(SEQ ID NO:225) BCDE rev: 5' CCT CCT GTT TCT CCG TAA GTG 3' (SEQ ID
NO:226) BCDEfor: 5' CAC TTA CGG AGA AAC AGG AGG 3' (SEQ ID NO:227)
hf01 DE-FG forward: 5' ACA GCT ACC ATC AGC GGC CTT AAA CCT GGC GTT
GAT TAT ACC ATC ACT GTG TAT GCT GTC ACT 3' (SEQ ID NO:228) Front FG
reverse: 5' AGT GAC AGC ATA CAC AGT 3' (SEQ ID NO:229) hf01 RT Flag
PolyA reverse: 5' TTT TTT TTT TTT TTT TTT TTA AAT AGC GGA TGC CTT
GTC GTC GTC GTC CTT GTA GTC TGT GCG GTA ATT AAT GGA 3' (SEQ ID
NO:230) 5-RI-hKDR-1B: 5' TAG AGA ATT CAT GGA GAG CAA GGT GCTG 3'
(SEQ ID NO:231) 3-EPO/hKDR-2312B: 5' AGG GAG AGC GTC AGG ATG AGT
TCC AAG TTC GTC TTT TCC 3' (SEQ ID NO:232) 5-RI-mKDR-1: 5' TAG AGA
ATT CAT GGA GAG CAA GGC GCT G 3' (SEQ ID NO:233) 3-EPO/mKDR-2312:
5' AGG GAG AGC GTC AGG ATG AGT TCC AAG TTG GTC TTT TCC 3' (SEQ ID
NO:234) 5-RI-hTrkB-1: 5' TAG AGA ATT CAT GAT GTC GTC CTG GAT AAG GT
3' (SEQ ID NO:235) 3-EpoR/hTrkB-1310: 5' AGG GAG AGC GTC AGG ATG
AGA TGT TCC CGA CCG GTT TTA 3' (SEQ ID NO:236) 5-hKDR/EPO-2274B: 5'
GGA AAA GAC GAA CTT GGA ACT CAT CCT GAC GCT CTC CCT 3' (SEQ ID
NO:237) 5-mKDR/EPO-2274: 5' GGA AAA GAC CAA CTT GGA ACT CAT CCT GAC
GCT CTC CCT 3' (SEQ ID NO:238) 3-XHO-EpoR-3066: 5' TAG ACT CGA GTC
AAG AGC AAG CCA CAT AGCT 3' (SEQ ID NO:239) 5'hTrkB/EpoR-1274: 5'
TAA AAC CGG TCG GGA ACA TCT CAT CCT GAC GCT CTC CCT 3' (SEQ ID
NO:240)
Buffers
[0231] The following buffers were utilized in the experiments
described herein. Buffer A (100 mM Tris HCl, 1M NaCl, 0.05%
Tween-20, pH 8.0); Buffer B (1.times.PBS, 0.02% Triton X100);
Buffer C (100 mM Tris HCl, 60 mM EDTA, 1M NaCl, 0.05% Triton X100,
pH 8.0); Buffer Ca (100 mM Tris HCl, 1M NaCl, 0.05% Triton X100, pH
8.0); Buffer D (2M NaCl, 0.05% Triton); Buffer E (1.times.PBS,
0.05% Triton X100, pH 7.4); Buffer F (1.times.PBS, 0.05% Triton
X100, 100 mM imidazole, pH 7.4); Buffer G (50 mM HEPES, 150 mM
NaCl, 0.02% TritonX-100, 1 mg/ml bovine serum albumin, 0.1 mg/ml
salmon sperm DNA, pH 7.4); Buffer H (50 mM HEPES, 150 mM NaCl,
0.02% TritonX-100, pH 7.4); Buffer I (1.times.PBS, 0.02%
TritonX-100, 1 mg/ml bovine serum albumin, 0.1 mg/ml salmon sperm
DNA, pH 7.4); Buffer J (1.times.PBS, 0.02% TritonX-100, pH 7.4);
Buffer K (50 mM NaH.sub.2PO.sub.4, 0.5 M NaCl, 5% glycerol, 5 mM
CHAPS, 25 mM imidazole, 1.times. Complete.TM. Protease Inhibitor
Cocktail (Roche), pH 8.0); Buffer L (50 mM NaH.sub.2PO.sub.4, 0.5 M
NaCl, 5% glycerol, 25 mM imidazole, pH 8.0); Buffer M (1.times.PBS,
pH 7.4, 25 mM imidazole, pH 7.4); Buffer N (1.times.PBS, 250 mM
imidazole, pH 7.4); Buffer 0 (10 mM HEPES, 150 mM NaCl, 0.005%
Tween 20, pH 7.4).
Primary Library Construction:
[0232] The construction of the library using the tenth domain of
human fibronectin as a scaffold was previously described (Xu et al,
2002, supra). Three loop regions, corresponding to positions 23-29,
52-55, and 77-86, respectively, were randomized using NNS (standard
nucleotide mixtures, where N=equimolar mixture of A, G, T, C;
S=equimolar mixture of G and C) as the coding scheme. Similar
libraries were constructed containing randomized regions only at
positions 23-29 and 77-86 (two loop library) or only at positions
77-86 (one loop library). These libraries were mixed in
approximately equimolar amounts. This mixed library contained
.about.1.times.10.sup.13 clones and was used in the KDR selection
that identified VR28.
Mutagenic Library Construction:
[0233] Hypermutagenic PCR. Scaffold mutation T(69)I in VR28 clone
was corrected back to wild type sequence by PCR (see below) and no
change in binding characteristics of VR28 binder to KDR was
observed. Mutations were introduced into the loop regions of VR28
using conditions described previously (Vartanian et al, Nuc. Acid
Res. 24:2627-2631, 1996). Three rounds of hypermutagenic PCR were
conducted on a VR28 template using primer pairs flanking each loop
(N-terminus forward/BCDE reverse, BCDE forward/DEFG reverse, DEFG
forward/C-terminus polyA). The resulting fragments were assembled
using overlap extension and PCR with flanking primers T7TMV Fn and
MK165-4 A20. DNA sequencing of the clones from the final PCR
reaction confirmed correct assembly of the library. Up to 30%
mutagenesis rate was observed in the loop regions, as compared to
1.5% in the scaffold regions.
[0234] Oligo mutagenesis. Oligo mutagenesis of the FG loop of VR28
by PCR utilized the VR28FG-50 primer, DEFG reverse primer and
flanking primers. At each nucleotide position encoding the FG loop,
primer VR28FG-50 contained 50% of the VR28 nucleotide and 50% of an
equimolar mixture of all four nucleotides (N) or of G or C(S). This
scheme was designed to result in approximately 67% of the amino
acids of the VR28 FG loop being randomly replaced by another amino
acid which was confirmed by DNA sequencing.
[0235] 159 (Q8L) randomized sub-libraries. Oligo mutagenesis of the
FG loop of Clone 159 (Q8L) clone, a three-step extension and
amplification was performed. For the first extension, pairs of
primers (a: F1U2/F2, b: F3159/F4, c: F5159/F6, d: F7X6159/F8) were
mixed in equal concentrations (100 pmol each) and amplified for 10
cycles. For the second extension, 1/20 of the first reactions were
combined (a/b and c/d) and amplification was continued for another
10 cycles. To bias the amplification in favor of extension rather
than re-annealing of fully complementary fragments, a linear
amplification of the half-construct products (0.5 pmol each) was
performed for an additional 20 cycles using 50 pmol of either F1U2
forward primer for fragment ab, or the C2asaiA20 reverse primer for
fragment cd. Finally, the extended half-construct fragments ab and
cd were combined and amplified for 20 cycles without any additional
components. Primer F7X6159 contained NNS at each of the first 6
coding positions of Clone 159 (Q8L) and was otherwise identical to
Clone 159 (Q8L). Correct assembly of the library 159 (Q8L)-FGX6 was
confirmed by DNA sequencing of clones from the final PCR reaction.
The sub-library contained .about.1.times.10.sup.9 clones.
[0236] For randomization of the DE loop of post round 6 (PR6)
selection pool of the 159 (Q8L)-FGX6 library, two half-construct
fragments were prepared by PCR using primers F1U2/F4 and
F5X5/C2asaiA20. The F5X5 primer contained NNS at the four positions
of the DE loop as well as at position 56. Then, the extended
fragments ab and cd were combined and amplified for 20 cycles
without any additional components.
[0237] Introduction of point mutations, deletion and random (NNS)
loop sequences into fibronectin-based scaffold proteins:
Scaffold mutation T(69)I of VR28 binder was corrected back to wild
type sequence in two-step PCR using VR28 clone as a template.
Half-construct fragments, obtained with primers N-terminus
forward/DEFG reverse and DEFG forward/C-terminus polyA, were
combined and the whole VR28 (169T) clone (designated as VR28 in the
text) was constructed using primers T7TMV Fn and MK165-4 A20.
Correction of N-terminus mutations in clone 159 (Q8 to L) was
performed by PCR with primers N-terminus forward/C-terminus polyA
followed by extension with primers T7TMV Fn and MK165-4 A20.
[0238] Introduction of deletion .DELTA.1-8 into the N-terminus of
fibronectin-based scaffold proteins was performed by amplification
using primers T7 TMV N-terminus deletion and MK165-4 A20.
[0239] Construction of the chimeras of E clones containing NNS loop
sequences was performed by two-step PCR. Loop regions were
amplified using primers T7 TMV N-terminus deletion/BCDE rev (a: BC
loop of E clones); N-terminus forward/hf01 BC reverse (b: BC NNS);
BCDE for/Front FG reverse (c: DE loop of E clones); BCDE for/hf01
DE reverse (d: DE NNS); hf01 DE-FG forward/hf01 RT-Flag PolyA
reverse (e: FG of E clones); hf01 DE-FG forward/hf01 FG reverse (f:
FG NNS). Fragments b/c/e, a/d/e, a/c/f were combined and the whole
pools were constructed by extension and amplification using primers
T7Tmv N-terminus deletion and hf01 RTFlag PolyA reverse.
[0240] All constructs were verified and/or analyzed by DNA
sequencing. All constructs and mutagenic libraries contained T7 TMV
promoter at the 5' flanking region and Flag tag or His.sub.6 tag
sequences at 3' flanking region for RNA-protein fusion production
and purification in vitro.
RNA-Protein Fusion Production
[0241] For each round of selection PCR DNA was transcribed using
MegaScript transcription kit (Ambion) at 37.degree. C. for 4 hours.
Template DNA was removed by DNase I (Ambion) digestion at
37.degree. C. for 20 minutes. RNA was purified by phenol/chloroform
extraction followed by gel filtration on a NAP-25 column
(Amersham). The puromycin linker PEG 6/10 (5' Pso u agc gga ugc XXX
XXX CC Pu 3', where Pso=C6-Psoralen, u,a,g,c=2'OMe-RNA, C=standard
amidities, X: Spacer Phosphoramidite 9
(9-O-Dimethoxytrityl-triethylene glycol,
1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite);
Pu=Puromycin-CPG) was synthesized as described previously (Kurz et
al, Nuc. Acid Res. 28:83, 2000). The linker was annealed to the
library RNA in 0.1 M NaCl, 25 mM Tris HCl, pH 7.0, by gradient
temperature decrease from 85.degree. C. to 4.degree. C. The linker
and RNA were then cross linked by exposing to UV light (365 nm) for
15 minutes. The cross-linked mixture (600 pmol RNA) was included in
an in vitro translation reaction using rabbit reticulocyte lysate
translation kit (Ambion) in the presence of .sup.35S-labeled
methionine at 30.degree. C. for 60 minutes. To enhance fusion
formation, 0.5 M KCl and 0.05 M MgCl.sub.2 were added to the
reaction and incubated for 30 minutes at 4.degree. C. Fusion
molecules were purified using oligo-dT cellulose (Sigma)
chromatography as follows. The translation and fusion mix was
diluted into buffer A (100 mM Tris HCl, 1M NaCl, 0.05% Tween-20, pH
8.0) and added to oligo dT cellulose. The slurry was rotated at
4.degree. C. for 1 hour and transferred to a spin column. Oligo dT
cellulose beads were washed on the column with 10 column volumes of
buffer A and eluted with 3 column volumes of H.sub.2O. Reverse
transcription reaction was conducted with SuperScript II Reverse
Transcription kit (Invitrogen) for 1 hour at 42.degree. C. using
primer Hu3'FLAGSTOP. To decrease potential non-specific binding
through reactive cysteines the thiol groups were reacted with 1 mM
of 2-nitro-5-thiocyanatobenzoic acid (NTCB) or N-ethylmaleimide
(NEM) alternatively over the course of the selection. The reaction
was carried out for 1 hour at room temperature. Fusion molecules
were further purified by anti-FLAG affinity chromatography using M2
agarose (Sigma). The M2 beads were added to the reaction and
rotated in buffer B (1.times.PBS, 0.02% Triton X100) for 1 hour at
4.degree. C. Then the beads were applied to a spin column, washed
with 5 column volumes of buffer B and fusion molecules were eluted
with 3 column volumes of 100 .mu.M Flag peptide DYKDDDDK (Sigma) in
buffer G. Fusion yield was calculated based on specific activity
measured by scintillation counting of .sup.35S-methionine in the
samples.
[0242] For the 159 (Q8L) randomized library, RNA-protein fusion was
prepared using a streamlined, semi-automated procedure in a
Kingfisher.TM. (ThermoLabSystems). The steps were similar to the
procedure described above except for several steps described below.
Purification of the RNA-protein fusion molecules was performed in
buffer C (100 mM Tris HCl, 60 mM EDTA, 1M NaCl, 0.05% Triton X100,
pH 8.0) on magnetic oligo dT beads (GenoVision). The beads were
washed with 10 reaction volumes of buffer Ca (100 mM Tris HCl, 1M
NaCl, 0.05% Triton X100, pH 8.0) and fusion proteins were eluted
with one volume of H.sub.2O. Reverse transcription (RT) was
conducted using primer C2RT. Fusion proteins were further purified
by His-tag affinity chromatography using Ni-NTA magnetic beads
(Qiagen). The RT reaction was incubated with Ni-NTA beads in buffer
D (2M NaCl, 0.05% Triton) for 20 minutes at room temperature, the
beads were then washed with 10 reaction volumes of buffer E
(1.times.PBS, 0.05% Triton X100, pH 7.4) and fusion molecules were
eluted with one volume of buffer F (1.times.PBS, 0.05% Triton X100,
100 mM imidazole, pH 7.4).
Selection:
[0243] Primary selection against KDR. Fusion library
(.about.10.sup.13 clones in 1 ml) was incubated with 150 .mu.l of
Protein A beads (Dynal) which was pre-immobilized with 200 nM of
human IgG1 for 1 hour at 30.degree. C. prior to selection to reduce
non-specific binding to both Protein A baeds and Fc protein
(preclear). The supernatant was then incubated in buffer G (50 mM
HEPES, 150 mM NaCl, 0.02% TritonX-100, 1 mg/ml bovine serum
albumin, 0.1 mg/ml salmon sperm DNA, pH 7.4) with KDR-Fc chimera
for 1 hour at 30.degree. C. with end-over-end rotation. Final
concentrations of KDR-Fc were 250 nM for Round 1, 100 nM for rounds
2-4 and 10 nM for rounds 5 and 6. The target was captured on 300
.mu.l of Protein A beads (Round 1) or 100 .mu.l of Protein A beads
(Rounds 2-6) for 30 minutes at 30.degree. C. with end-over-end
rotation and beads were washed 5 times with 1 ml of buffer G (50 mM
HEPES, 150 mM NaCl, 0.02% TritonX-100, pH 7.4). Bound fusion
molecules were eluted with 100 .mu.l of 0.1 M KOH into 50 .mu.l of
1 M Tris HCl, pH 8.0. DNA was amplified from elution by PCR using
flanking primers T7TMV Fn and MK165-4 A20.
[0244] Affinity and specificity maturation of KDR binder VR28.
Clone VR28 was mutagenized by hypermutagenic PCR or oligo-directed
mutagenesis as described above and fusion sub-libraries were
constructed. Following pre-clear with Protein A beads selection was
performed in buffer I (1.times.PBS, 0.02% TritonX-100, 1 mg/ml
bovine serum albumin, 0.1 mg/ml salmon sperm DNA, pH 7.4) for four
rounds according to procedure described above. DNA was amplified
from elution by PCR using primers T7TMV Fn and MK165-4 A20. Lower
target concentrations (0.1 nM KDR for first four rounds of
selection) were used for libraries derived from oligo mutagenesis
and then 1 nM mouse VEGF-R2 (Flk-1) was introduced for three
additional rounds of selection. Primers T7 TMV N-terminus deletion
and MK165-4 A20 were used for PCR in the last 3 rounds. For
specificity maturation of KDR binder 159 first 6 positions of the
FG loop of clone 159 Q(8)L were randomized by PCR as described
above. Binding of the fusion sub-library to biotinylated mouse
VEGF-R2 (70 nM) was performed in buffer I at room temperature for
30 minutes. The rest of the selection procedure was continued in
Kingfisher.TM. (ThermoLabSystems). The biotinylated target was
captured on 50 .mu.l of streptavidin-coated magnetic beads (Dynal)
and the beads were washed with 10 volumes of buffer I and one
volume of buffer J (1.times.PBS, 0.02% TritonX-100, pH 7.4). Bound
fusion molecules were eluted with 100 .mu.l of 0.1 M KOH into 5.01
of 1 M Tris HCl, pH 8.0. DNA was amplified from elution by PCR
using primers F1U2 and C2asaiA20. After four rounds of selection an
off-rate/rebinding selection against 7 nM Flk-1 was applied for
another two rounds as follows. After the binding reaction with
biotinylated mouse Flk-1 had progressed for 30 minutes, a 100-fold
excess of non-biotinylated Flk-1 was added and the reaction
continued for another 6 hours to allow time for the weak binders to
dissociate. The biotinylated target was captured on 50 .mu.l of
streptavidin beads (Dynal) and beads were washed 5 times with 1 ml
of buffer J. Bound fusion molecules were eluted by incubation at
75.degree. C. for 5 minutes. Supernatant was subjected to
re-binding to 7 nM Flk-1 and standard selection procedure was
continued. DNA from the final elution pool was subjected to DE loop
randomization (see above) and fusion sub-library was selected
against 7 nM mouse VEGF-R2 for three rounds. At the fourth round an
off-rate selection was applied with re-binding to 1 nM human
VEGF-R2. Final DNA was amplified from elution by PCR using primers
F1U2 and C2asaiA20.
Radioactive Equilibrium Binding Assay
[0245] To prepare .sup.35S-labeled binding proteins for analysis,
mRNA was prepared as described above for RNA-protein fusion
production but the linker ligation step was omitted. The mRNA was
expressed using rabbit reticulocyte lysate translation kit (Ambion)
in the presence of .sup.35S-labeled Met at 30.degree. C. for 1
hour. Expressed protein was purified on M2-agarose Flag beads
(Sigma) as described above. This procedure produced the encoded
protein without the nucleic acid tail. In a direct binding assay,
VEGF-R2-Fc fusions in concentrations ranging from 0 to 200 nM were
added to a constant concentration of the purified protein (0.2 or
0.5 nM) and incubated at 30.degree. C. for 1 hour in buffer B. The
receptor-binder complexes were captured using Protein A magnetic
beads for another 10 minutes at room temperature using a
Kingfisher.TM.. The beads were washed with six reaction volumes of
buffer B. The protein was eluted from the beads with 100 .mu.L of
0.1 M KOH. 50 .mu.L of the reaction mixture and elution were dried
onto a LumaPlate-96 (Packard) and the amount of .sup.35S on the
plate was measured using a TopCount NXT instrument (Packard). The
amount of fibronectin-based scaffold protein bound to the target
was estimated as a percent of radioactivity eluted from Protein A
magnetic beads compare to radioactivity in the reaction mixture.
Nonspecific binding of fibronectin-based scaffold proteins to the
beads was determined by measuring binding in the absence of KDR-Fc
and represented less than 1-2% of the input. Specific binding was
obtained through subtraction of nonspecific binding from total
binding. Data was analyzed using the GraphPad Prizm software
(GraphPad Software, Inc, San Diego, Calif.), fitted using a one
site, non-linear binding equation.
Expression and Purification of Soluble Fibronectin-Based Scaffold
Protein Binders:
[0246] For expression in E. coli residues 1-101 of each clone
followed by the His.sub.6 tag were cloned into a pET9d-derived
vector and expressed in E. coli BL21 (DE3) pLysS cells
(Invitrogen). 20 ml of overnight culture was used to inoculate 1
liter of LB medium containing 50 .mu.g/mL kanamycin and 34 .mu.g/mL
chloromphenicol. The culture was grown at 37.degree. C. until
A.sub.600 0.4-0.6. After induction with 1 mM
isopropyl-.beta.-thiogalactoside (IPTG, Invitrogen) the culture was
grown for another 3 hours at 37.degree. C. and harvested by
centrifugation for 30 minutes at 3,000 g at 4.degree. C. The cell
pellet was resuspended in 50 mL of lysis buffer K (50 mM
NaH.sub.2PO.sub.4, 0.5 M NaCl, 5% glycerol, 5 mM CHAPS, 25 mM
imidazole, 1.times. Complete.TM. Protease Inhibitor Cocktail
(Roche), pH 8.0) Buffer L and sonicated on ice at 80 W for four 15
second pulses separated by ten-second pauses. The soluble fraction
was separated by centrifugation for 30 minutes at 30,000 g at
4.degree. C. The supernatant was rotated for 1 hour at 4.degree. C.
with 10 mL of TALON.TM. Superflow.TM. Metal Affinity Resin
(Clontech) pre-equilibrated with wash buffer L (50 mM
NaH.sub.2PO.sub.4, 0.5 M NaCl, 5% glycerol, 25 mM imidazole, pH
8.0). The resin was then washed with 10 column volumes of buffer L
and 30 column volumes of buffer M (1.times.PBS, pH 7.4, 25 mM
imidazole, pH 7.4). Protein was eluted with 5 column volumes of
buffer N (1.times.PBS, 250 mM imidazole, pH 7.4) and dialyzed
against 1.times.PBS at 4.degree. C. Any precipitate was removed by
filtering at 0.22 .mu.m (Millipore).
BIAcore Analysis of the Soluble Fibronectin-Based Scaffold
Proteins:
[0247] The binding kinetics of fibronectin-based scaffold proteins
binding proteins to the target was measured using BIAcore 2000
biosensor (Pharmacia Biosensor). Human and mouse VEGF-R2-Fc fusions
were immobilized onto a CM5 sensor chip and soluble binding
proteins were injected at concentrations ranging from 0 to 100 nM
in buffer 0 (10 mM HEPES, 150 mM NaCl, 0.005% Tween 20, pH 7.4).
Sensorgrams were obtained at each concentration and were evaluated
using a program, BIA Evaluation 2.0 (BIAcore), to determine the
rate constants k.sub.a (k.sub.on) and k.sub.d (k.sub.off) The
affinity constant, K.sub.D was calculated from the ratio of rate
constants k.sub.off/k.sub.on.
[0248] For inhibition experiments, human VEGF.sub.165 was
immobilized on a surface of CM-5 chip and KDR-Fc was injected at a
concentration of 20 nM in the presence of different concentrations
of soluble binding proteins ranging from 0 to 100 nM. IC.sub.50 was
determined at a concentration when only 50% of KDR-Fc binding to
the chip was observed.
Reversible Refolding of a VEGFR Binding Polypeptide:
[0249] Differential scanning calorimetry (DSC) analysis was
performed on M5FL protein in 100 mM sodium acetate buffer (pH 4.5).
An initial DSC run (Scan 1) was performed in a N-DSC II calorimeter
(Calorimetry Sciences Corp) by ramping the temperature from
5-95.degree. C. at a rate of 1 degree per minute, followed by a
reverse scan (not shown) back to 10 degrees, followed by a second
run (Scan 2). Under these conditions, data were best fit using a
two transition model (Tm=77.degree. C. and 67.degree. C. using
Orgin software (OrginLab Corp)). See FIG. 10.
PEGylation of the M5FL Protein:
[0250] The C100-form of the M5FL protein, which has the complete
sequence of M5FL with the Ser at position 100 mutated to a Cysteine
including the additional C-terminal His-tag used to purify the
protein. The purified M5FL-C100 protein was modified at the single
cysteine residue by conjugating various maleimide-derivatized PEG
forms (Shearwater). The resulting reacted proteins were run on a
4-12% polyacrylamide gel (FIG. 11).
Construction of Cell Lines:
[0251] Plasmid construction. Plasmids, encoding chimeric receptors
composed of the transmembrane and cytoplasmic domains of the human
erythropoietin receptor (EpoR) fused to the extracellular domains
of KDR, Flk-1, or human TrkB were constructed by a two-step PCR
procedure. PCR products encoding the extracellular domains were
amplified from plasmids encoding the entire receptor gene: KDR
(amino acids 1 to 764) was derived from clone PR1371_H11 (OriGene
Technologies, Rockville, Md.) with primers
5-RI-hKDR-1B/3-EPO/hKDR-2312B, flk-1 (amino acids 1 to 762) was
derived from clone #4238984 (IMAGE) with primers
5-RI-mKDR-1/3-EPO/mKDR-2312, and human TrkB (from amino acids 1 to
430) from clone #X75958 (Invitrogen Genestorm) with primers
5-RI-hTrkB-1/3-EpoR/hTrkB-1310. PCR products encoding the EpoR
transmembrane and cytoplasmic domains (amino acids 251 to 508) were
amplified from clone #M60459 (Invitrogen Genestorm) with the common
primer 3-XHO-EpoR-3066 and one of three gene-specific primers
5-hKDR/EPO-2274B (KDR), 5-mKDR/EPO-2274 (flk-1), and
5'hTrkB/EpoR-1274 (human TrkB), which added a short sequence
complementary to the end of the receptor fragment PCR product.
Second, PCR products encoding the two halves of the chimeric genes
were mixed and amplified with 3-XHO-EpoR-3066 and the 5' primers
(5-RI-hKDR-1B, 5-RI-mKDR-1, and 5-RI-hTrkB-1) specific for each
gene used in the previous cycle of amplification. The resulting PCR
products were digested with EcoRI and XhoI and cloned into
pcDNA3.1(+) (Invitrogen) to generate the plasmids phKE8 (human
KDR/EpoR fusion), pmKE2 (flk-1/EpoR fusion), and phTE (TrkB/EpoR
fusion).
[0252] Construction of cell lines for flow cytometry. CHO-K1 cells
(American Type Culture Collection, Manassas, Va.) were stably
transfected using Lipofectamine 2000 (Invitrogen) with either pcDNA
3.1 (Invitrogen) alone, pmKE2 alone, or a mixture of pcDNA 3.1 and
a plasmid encoding full-length human KDR (Origene Inc., clone
PR1371-H11). Stable transfectants were selected and maintained in
the presence of 0.5 mg/ml of Geneticin (Invitrogen). The human
KDR-expressing clone designated CHO-KDR and the murine
VEGFR-2/EpoR-chimera-expressing population designated CHO-Flk were
obtained by fluorescence activated cell sorting of the transfected
population following staining with an anti-KDR polyclonal antiserum
(R&D Systems). CHO-KDR and CHO-Flk cell lines were grown
routinely in Dulbecco's modified Eagle's medium (DMEM; Gibco)
supplemented with 10% (v/v) fetal bovine serum (FBS), 0.5 mg/ml
Geneticin, 100 U/ml penicillin, 0.25 .mu.g/ml amphotericin B, 100
.mu.g/ml streptomycin and 2 mM L-glutamine.
[0253] Construction of Ba/F3 cell lines. Cell lines that would
proliferate in response to VEGF binding by VEGFR-2 were constructed
by transfection of the murine pre-B cell line Ba/F3 (DSMZ,
Braunschweig, Germany) with phKE8 or pmKE2, receptor chimeras
consisting of the extracellular domains of human or murine VEGFR-2
fused to the transmembrane and cytoplasmic domains of the human
erythropoietin receptor (see above). Ba/F3 cells were maintained in
minimal Ba/F3 medium (RPMI-1640 (Gibco) containing 10% FBS, 100
U/ml penicillin, 0.25 .mu.g/ml amphotericin B, 100 .mu.g/ml
streptomycin and 2 mM L-glutamine) supplemented with 10%
conditioned medium from WEHI-3B cells (DSMZ; grown in Iscove's
modified Dulbecco's medium (Gibco)/10% FBS/25 .mu.M
.beta.-mercaptoethanol) as a source of essential growth factors.
Following electroporation with the plasmids pmKE2 or phKE8, stable
transfectants were selected in 0.75 mg/ml Geneticin.
Geneticin-resistant populations were transferred to minimal Ba/F3
medium containing 100 ng/ml of human VEGF.sub.165 (R&D
Systems), and the resulting VEGF-dependent populations were
designated Ba/F3-Flk and Ba/F3-KDR. Control cell line expressing a
chimeric TrkB receptor (Ba/F3-TrkB) that would be responsive to
stimulation by NT-4, the natural ligand for TrkB was similarly
constructed using the plasmid phTE and human NT-4 (R&D
Systems).
Analysis of Cell Surface Binding of Fibronectin-Based Scaffold
Proteins:
[0254] Binding of fibronectin-based scaffold protein to
cell-surface KDR and Flk-1 was analyzed simultaneously on
VEGF-R2-expressing and control cells by flow cytometry. CHO-pcDNA3
cells (control) were released from their dishes with trypsin-EDTA,
washed in Dulbecco's PBS without calcium and magnesium
(D-PBS.sup.-; Invitrogen), and stained for 30 minutes at 37.degree.
C. with 1.5 .mu.M CMTMR
(5-(and-6)-(((4-chloromethyl)benzoyl)amino)-tetramethylrhodamine)
(Molecular Probes). The cells were washed in D-PBS.sup.- and
incubated for a further 30 minutes at 37.degree. C., and then
resuspended in blocking buffer (D-PBS.sup.-/10% fetal bovine serum)
on ice. CHO-KDR or CHO-Flk cells were treated identically except
that CMTMR was omitted. 75,000 of CMTMR-stained CHO-pcDNA3 cells
were mixed with an equal number of unstained CHO-KDR or CHO-Flk
cells in each well of a V-bottom 96-well plate. All antibodies and
fibronectin-based scaffold proteins were diluted in 25 .mu.l/well
of blocking buffer, and each treatment was conducted for 1 hour at
4.degree. C. Cell mixtures were stained with His.sub.6-tagged
fibronectin-based scaffold proteins, washed twice with cold
D-PBS.sup.-, and then treated with 2.5 .mu.g/ml anti-His.sub.6 MAb
(R&D Systems), washed, and stained with 4 .mu.g/ml Alexa Fluor
488-conjugated anti-mouse antibody (Molecular Probes). For cells
treated with an anti-KDR mouse monoclonal antibody (Accurate
Chemical, Westbury, N.Y.) or an anti-flk-1 goat polyclonal antibody
(R&D Systems), the anti-His.sub.6 step was omitted, and
antibody binding was detected with the species-appropriate Alexa
Fluor 488 conjugated secondary antibody (Molecular Probes).
Following staining, cells were resuspended in 200 .mu.l/well
D-PBS.sup.-/1% FBS/1 .mu.g/ml 7-aminoactinomycin D (7-AAD;
Molecular Probes) and analyzed by flow cytometry on a FACSCalibur
(Becton Dickinson, San Jose, Calif.) equipped with a 488 nM laser.
Following gating to exclude dead cells (7-AAD positive),
VEGFR-2-expressing cells and CHO-pcDNA3 cells were measured
independently for Alexa Fluor 488 fluorescence by gating on the
CMTMR-negative or -positive populations, respectively. Control
experiments showed that staining with CMTMR did not interfere with
the detection of Alexa Fluor 488-conjugated antibodies on the
surface of the stained cells.
[0255] Cell-surface binding was also assessed by fluorescence
microscopy using the secondary antibodies described above. For
these studies, antibodies were diluted in D-PBS containing calcium
and magnesium (D-PBS.sup.+)/10% FBS. Cells were grown on 24- or
96-well plates, and following staining were kept in D-PBS.sup.+ for
observation on an inverted fluorescence microscope.
Ba/F3 Cell Proliferation Assay:
[0256] Ba/F3 cells were washed three times in minimal Ba/F3 medium
and resuspended in the same medium containing 15.8 ng/ml of
proliferation factor (human VEGF.sub.165, murine VEGF.sub.164, or
hNT-4 for Ba/F3-KDR, Ba/F3-Flk, or Ba/F3-TrkB cells, respectively),
and 95 .mu.l containing 5.times.10.sup.4 Ba/F3-KDR cells or
2.times.10.sup.4 Ba/F3-Flk or Ba/F3-TrkB cells were added per well
to a 96-well tissue culture plate. 5 .mu.l of serial dilutions of
test protein in PBS was added to each well for a final volume of
100 .mu.l Ba/F3 medium/5% PBS/15 ng/ml growth factor. After
incubation for 72 hours at 37.degree. C., proliferation was
measured by addition of 20 .mu.l of CellTiter 96.RTM. Aqueous One
Solution Reagent (Promega) to each well, incubation for 4 hours at
37.degree. C., and measurement of the absorbance at 490 nm using a
microtiter plate reader (Molecular Dynamics).
HUVEC Cell Proliferation Assay:
[0257] HUVEC cells (Clonetics, Walkersville, Md.) from passage 2-6
were grown in EGM-2 medium (Clonetics). 5000 cells/well were
resuspended in 200 .mu.l starvation medium (equal volumes of DMEM
(Gibco) and F-12K medium (ATCC), supplemented with 0.2% fetal
bovine serum and 1.times. penicillin/streptomycin/fungizone
solution (Gibco)), plated in 96-well tissue culture plates and
incubated for 48 hours. Fibronectin-based binding proteins were
added to the wells and incubated for 1 hour at 37.degree., and then
human VEGF.sub.165 was added to a final concentration of 16 ng/ml.
After 48 hours incubation, cell viability was measured by addition
of 30 .mu.l/well of a mixture of 1.9 mg/ml CellTiter96.RTM. AQueous
MTS reagent (Promega) with 44 .mu.g/ml phenazine methosulfate
(Sigma) and measurement of absorbance at 490 nm as described above
for Ba/F3 cells.
Example 12
Antibody Light Chain-Based VEGFR Binding Polypeptides
[0258] FIGS. 21A and 21B show amino acid sequences of VEGFR binding
polypeptides (SEQ ID NOs:241-310) based on an antibody light chain
variable region (VL) framework/scaffold.
[0259] Light chain variable domain proteins were generated using
the PROfusion.TM. system, as described above for use with
.sup.10Fn3-derived proteins.
[0260] All references cited herein are hereby incorporated by
reference in their entirety.
TABLE-US-00016 TABLE 1 Preferred Specific Peptide Sequences SEQ
Binding Kd Binding Kd ID Clone N- DE to 1 nM KDR, to 1 nM FLK, NO
Name terminus BC Loop Loop FG Loop KDR, % nM FLK, % nM KDR Binders
6 K1 Del 1-8 RHPHFPTR LQPPT M G L Y G H E L L T P 48 0.55 7 K2 Del
1-8 RHPHFPTR LQPPT D G E N G Q F L L V P 48 1.19 8 K5 Del 1-8
RHPHFPTR LQPPT M G P N D N E L L T P 47 1.54 9 K3 Del 1-8 RHPHFPTR
LQPPT A G W D D H E L F I P 45 1.15 10 K7 Del 1-8 RHPHFPTR LQPPT S
G H N D H M L M I P 40 2.2 11 K4 Del 1-8 RHPHFPTR LQPPT A G Y N D Q
I L M T P 38 1.95 12 K9 Del 1-8 RHPHFPTR LQPPT F G L Y G K E L L I
P 35 1.8 13 K10 Del 1-8 RHPHFPTR LQPPT T G P N D R L L F V P 33
0.57 14 K12 Del 1-8 RHPHFPTR LQPPT D V Y N D H E I K T P 29 0.62 15
K6 Del 1-8 RHPHFPTR LQPPT D G K D G R V L L T P 27 0.93 16 K15 Del
1-8 RHPHFPTR LQPPT E V H H D R E I K T P 25 0.35 17 K11 Del 1-8
RHPHFPTR LQPPT Q A P N D R V L Y T P 24 1.16 18 K14 Del 1-8
RHPHFPTR LQPPT R E E N D H E L L I P 20 0.57 19 K8 Del 1-8 RHPHFPTR
LQPPT V T H N G H P L M T P 18 3.3 20 K13 Del 1-8 RHPHFPTR LQPPT L
A L K G H E L L T P 17 0.58 21 VR28 WT RHPHFPTR LQPPT V A Q N D H E
L I T P 3 11 22 159 WT RHPHFPTR LQPPA M A Q S G H E L F T P KDR and
FLK Binders 24 E29 Del 1-8 RHPHFPTR LQPPT V E R N G R V L M T P 41
44 1.51 0.91 25 E19 Del 1-8 RHPHFPTR LQPPT V E R N G R H L M T P 38
40 1.3 0.66 33 E25 Del 1-8 RHPHFPTR LQPPT L E R N G R E L M T P 41
28 1.58 1.3 45 E9 Del 1-8 RHPHFPTR LQPPT E E R N G R T L R T P 24
34 2.37 1.4 50 E24 Del 1-8 RHPHFPTR LQPPT V E R N D R V L F T P 24
29 54 E26 Del 1-8 RHPHFPTR LQPPT V E R N G R E L M T P 27 20 1.66
2.05 59 E28 Del 1-8 RHPHFPTR LQPPT L E R N G R E L M V P 19 21 1.63
2.1 60 E3 Del 1-8 RHPHFPTR LQPPT D G R N D R K L M V P 37 14 0.96
5.4 65 E5 Del 1-8 RHPHFPTR LQPPT D G Q N G R L L N V P 26 10 0.4
3.2 91 E23 Del 1-8 RHHPHFPTR LQPPT V H W N G R E L M T P 36 7 92 E8
Del 1-8 RHPHFPTR LQPPT E E W N G R V L M T P 51 10 93 E27 Del 1-8
RHPHFPTR LQPPT V E R N G H T L M T P 37 9 94 E16 Del 1-8 RHPHFPTR
LQPPT V E E N G R Q L M T P 35 0 95 E14 Del 1-8 RHPHFPTR LQPPT L E
R N G Q V L F T P 33 11 96 E20 Del 1-8 RHPHFPTR LQPPT V E R N G Q V
L Y T P 43 11 97 E21 Del 1-8 RHPHFPTR LQPPT W G Y K D H E L L I P
47 1 98 E22 Del 1-8 RHPHFPTR LQPPT L G R N D R E L L T P 45 3 99 E2
Del 1-8 RHPHFPTR LQPPT D G P N D R L L N I P 53 10 100 E12 Del 1-8
RHPHFPTR LQPPT F A R D G H E I L T P 36 1 101 E13 Del 1-8 RHPHFPTR
LQPPT L E Q N G R E L M T P 38 1 102 E17 Del 1-8 RHPHFPTR LQPPT V E
E N G R V L N T P 32 10 103 E15 Del 1-8 RHPHFPTR LQPPT L E P N G R
Y L M V P 52 2 104 E10 Del 1-8 RHPHFPTR LQPPT E G R N G R E L F I P
53 3 154 M2 WT RHPHFPTR LQPPA W E R N G R E L F T P 156 M3 WT
RHPHFPTR LQPPA K E R N G R E L F T P 172 M4 WT RHPHFPTH LQPPA T E R
T G R E L F T P 173 M8 WT RHPHFPTH LQPPA K E R S G R E L F T P 175
M6 WT RHPHFPTH LQPPA L E R D G R E L F T P 180 M7 WT RHPHFPTR LQPTT
W E R N G R E L F T P 181 M1 WT RHPHFPTR LQPTV L E R N D R E L F T
P 177 M5FL WT RHPHFPTR LQPPL K E R N G R E L F T P
TABLE-US-00017 TABLE 2 KDR & FLK binders SEQ ID DE NO Clone
Name N-terminus N-Terminus Framework 1 BC Loop Framework 2 Loop 23
D12 Del 1-8 EVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT 24
E29 Del 1-8 EVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT 25
E19 Del 1-8 EVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT 26
D1 Del 1-8 EVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT 27
C6 Del 1-8 EVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT 28
EGE5 Del 1-8 EVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT 29
EGE2 Del 1-8 EVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT 30
D4 Del 1-8 EVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT 31
E25 Del 1-8 EVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT 32
EGE6 Del 1-8 EVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT 33
C7 Del 1-8 EVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT 34
D9 Del 1-8 EVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT 35
EGE3 Del 1-8 EVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT 36
D3 Del 1-8 EVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT 37
D2 Del 1-8 EVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT 38
C8 Del 1-8 EVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT 39
EGE4 Del 1-8 EVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT 40
D7 Del 1-8 EVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT 41
D5 Del 1-8 EVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT 42
B3 Del 1-8 EVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT 43
E9 Del 1-8 EVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT 44
D6 Del 1-8 EVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT 45
EGE7 Del 1-8 EVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT 46
EGE1 Del 1-8 EVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT 47
F9 Del 1-8 EVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT 48
E24 Del 1-8 EVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT 49
B11 Del 1-8 EVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT 50
B12 Del 1-8 EVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT 51
B5 Del 1-8 EVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT 52
E26 Del 1-8 EVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT 53
C12 Del 1-8 EVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT 54
F4 Del 1-8 EVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT 55
E18 Del 1-8 EVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT 56
C11 Del 1-8 EVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT 57
E28 Del 1-8 EVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT 58
E3 Del 1-8 EVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT 59
F8 Del 1-8 EVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT 60
F3 Del 1-8 EVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT 61
B10 Del 1-8 EVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT 62
E6 Del 1-8 EVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT 63
E5 Del 1-8 EVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT 64
G4 Del 1-8 EVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT 65
A3 Del 1-8 EVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT 66
A4 Del 1-8 EVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT 67
A6 Del 1-8 EVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT 68
A7 Del 1-8 EVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT 69
A8 Del 1-8 EVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT 70
A9 Del 1-8 EVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT 71
A10 Del 1-8 EVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT 72
EGE11 Del 1-8 EVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT
73 A11 Del 1-8 EVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT
74 A12 Del 1-8 EVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT
75 B4 Del 1-8 EVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT
76 B6 Del 1-8 EVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT
77 B7, B8 Del 1-8 EVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP
LQPPT 78 B11 Del 1-8 EVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP
LQPPT 79 C1 Del 1-8 EVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP
LQPPT 80 C2 Del 1-8 EVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP
LQPPT 81 C3 Del 1-8 EVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP
LQPPT 82 C9 Del 1-8 EVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP
LQPPT 83 C10 Del 1-8 EVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP
LQPPT 84 D11 Del 1-8 EVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP
LQPPT 85 EGE8 Del 1-8 EVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP
LQPPT 86 EGE9 Del 1-8 EVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP
LQPPT 87 EGE10 Del 1-8 EVVAATPTSLLISW RHPHFPTR
YYRITYGETGGNSPVQEFTVP LQPPT 88 EGE11 Del 1-8 EVVAATPTSLLISW
RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT 89 E23 Del 1-8 EVVAATPTSLLISW
RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT 90 E8 Del 1-8 EVVAATPTSLLISW
RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT 91 E27 Del 1-8 EVVAATPTSLLISW
RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT 92 E16 Del 1-8 EVVAATPTSLLISW
RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT 93 E14 Del 1-8 EVVAATPTSLLISW
RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT 94 E20 Del 1-8 EVVAATPTSLLISW
RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT 95 E21 Del 1-8 EVVAATPTSLLISW
RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT 96 E22 Del 1-8 EVVAATPTSLLISW
RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT 97 E2 Del 1-8 EVVAATPTSLLISW
RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT 98 E12 Del 1-8 EVVAATPTSLLISW
RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT 99 E13 Del 1-8 EVVAATPTSLLISW
RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT 100 E17 Del 1-8 EVVAATPTSLLISW
RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT 101 E15 Del 1-8 EVVAATPTSLLISW
RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT 102 E10 Del 1-8 EVVAATPTSLLISW
RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT 103 F1 Del 1-8 EVVAATPTSLLISW
RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT 104 F5 Del 1-8 EVVAATPTSLLISW
RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT 105 F6 Del 1-8 EVVAATPTSLLISW
RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT 106 F7 Del 1-8 EVVAATPTSLLISW
RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT 107 F10 Del 1-8 EVVAATPTSLLISW
RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT 108 F11 Del 1-8 EVVAATPTSLLISW
RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT 109 F12 Del 1-8 EVVAATPTSLLISW
RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT 110 G1 Del 1-8 EVVAATPTSLLISW
RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT 111 G2 Del 1-8 EVVAATPTSLLISW
RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT 112 G3 Del 1-8 EVVAATPTSLLISW
RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT 113 MWF10 WT
VSDVPRDLEVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPA 114
MWA10 WT VSDVPRDLEVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP
LQPPA 115 MWA2 WT VSDVPRDLEVVAATPTSLLISW RHPHFPTR
YYRITYGETGGNSPVQEFTVP LQPPA 116 MWC10 WT VSDVPRDLEVVAATPTSLLISW
RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPA 117 MWB7 WT
VSDVPRDLEVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPA 118
MWH8 WT VSDVPRDLEVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPA
119 MWA10 WT VSDVPRDLEVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP
LQPPA 120 MWB2 WT VSDVPRDLEVVAATPTSLLISW RHPHFPTR
YYRITYGETGGNSPVQEFTVP LQPPA 121 MWC3-f1 WT VSDVPRDLEVVAATPTSLLISW
RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPA 122 MWG11 WT
VSDVPRDLEVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPA 123
MWG11 WT VSDVPRDLEVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP
LQPPA 124 MWD3-f1 WT VSDVPRDLEVVAATPTSLLISW RHPHFPTR
YYRITYGETGGNSPVQEFTVP LQPPA 125 MWE11 WT VSDVPRDLEVVAATPTSLLISW
RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPA 126 MWD10 WT
VSDVPRDLEVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPA 127
MWC1 WT VSDVPRDLEVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPA
128 MWA12 WT VSDVPRDLEVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP
LQPPA 129 MWB3-f1 WT VSDVPRDLEVVAATPTSLLISW RHPHFPTR
YYRITYGETGGNSPVQEFTVP LQPPA 130 MWA11 WT VSDVPRDLEVVAATPTSLLISW
RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPA 131 MWG12 WT
VSDVPRDLEVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPA 132
MWH11 WT VSDVPRDLEVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP
LQPPA 133 MWD12 WT VSDVPRDLEVVAATPTSLLISW RHPHFPTR
YYRITYGETGGNSPVQEFTVP LQPPA 134 MWH5 WT VSDVPRDLEVVAATPTSLLISW
RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPA 135 MWA1 WT
VSDVPRDLEVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPA 136
MWG4-f1 WT VSDVPRDLEVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP
LQPPA 137 MWA12 WT VSDVPRDLEVVAATPTSLLISW RHPHFPTR
YYRITYGETGGNSPVQEFTVP LQPPA 138 MWG11 WT VSDVPRDLEVVAATPTSLLISW
RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPA 139 MWC12 WT
VSDVPRDLEVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPA 140
MWF11 WT VSDVPRDLEVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP
LQPPA 141 MWE11 WT VSDVPRDLEVVAATPTSLLISW RHPHFPTR
YYRITYGETGGNSPVQEFTVP LQPPA 142 MWD10 WT VSDVPRDLEVVAATPTSLLISW
RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPA
143 MWC4-f1 WT VSDVPRDLEVVAATPTSLLISW RHPHFPTR
YYRITYGETGGNSPVQEFTVP LQPPA 144 MWF3 WT VSDVPRDLEVVAATPTSLLISW
RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPA 145 MWB2 WT
VSDVPRDLEVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPA 146
MWE10 WT VSDVPRDLEVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP
LQPPA 147 MWD9 WT VSDVPRDLEVVAATPTSLLISW RHPHFPTR
YYRITYGETGGNSPVQEFTVP LQPPA 148 MWH3-f1 WT VSDVPRDLEVVAATPTSLLISW
RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPA 149 MWG10 WT
VSDVPRDLEVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPA 150
MWH11 WT VSDVPRDLEVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP
LQPPA 151 MWF11 WT VSDVPRDLEVVAATPTSLLISW RHPHFPTR
YYRITYGETGGNSPVQEFTVP LQPPA 152 M2 WT VSDVPRDLEVVAATPTSLLISW
RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPA 153 MWB09-f1 WT
VSDVPRDLEVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPA 154 M3
WT VSDVPRDLEVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPA 155
MWA3 WT VSDVPRDLEVVAATPTSLLISW LHPHFPTH YYRITYGETGGNSPVQEFTVP LQPPA
156 MWE10 WT VSDVPRDLEVVAATPTSLLISW RHPHFPTH YYRITYGETGGNSPVQEFTVP
LQPPA 157 MWG3 WT VSDVPRDLEVVAATPTSLLISW LHPHFPTH
YYRITYGETGGNSPVQEFTVP LQPPA 158 MWD5 WT VSDVPRDLEVVAATPTSLLISW
RHPHFPTH YYRITYGETGGNSPVQEFTVP LQPPA 159 MWC3 WT
VSDVPRDLEVVAATPTSLLISW LHPHFPTH YYRITYGETGGNSPVQEFTVP LQPPA 160
MWH3 WT VSDVPRDLEVVAATPTSLLISW LHPHFPTH YYRITYGETGGNSPVQEFTVP LQPPA
161 MWC2 WT VSDVPRDLEVVAATPTSLLISW LHPHFPTH YYRITYGETGGNSPVQEFTVP
LQPPA 162 MWE2 WT VSDVPRDLEVVAATPTSLLISW LHPHFPTH
YYRITYGETGGNSPVQEFTVP LQPPA 163 MWA2 WT VSDVPRDLEVVAATPTSLLISW
FHPHFPTH YYRITYGETGGNSPVQEFTVP LQPPA 164 MWD3 WT
VSDVPRDLEVVAATPTSLLISW LHPHFPTH YYRITYGETGGNSPVQEFTVP LQPPA 165
MWE3 WT VSDVPRDLEVVAATPTSLLISW LHPHFPTH YYRITYGETGGNSPVQEFTVP LQPPA
166 MWB3 WT VSDVPRDLEVVAATPTSLLISW FHPHFPTH YYRITYGETGGNSPVQEFTVP
LQPPA 167 MWD2 WT VSDVPRDLEVVAATPTSLLISW LHPHFPTH
YYRITYGETGGNSPVQEFTVP LQPPA 168 MWC11 WT VSDVPRDLEVVAATPTSLLISW
RHPHFPTH YYRITYGETGGNSPVQEFTVP LQPPA 169 MWH12 WT
VSDVPRDLEVVAATPTSLLISW RHPHFPTH YYRITYGETGGNSPVQEFTVP LQPPA 170 M4
WT VSDVPRDLEVVAATPTSLLISW RHPHFPTH YYRITYGETGGNSPVQEFTVP LQPPA 171
M8 WT VSDVPRDLEVVAATPTSLLISW RHPHFPTH YYRITYGETGGNSPVQEFTVP LQPPA
172 MWF10-f1 WT VSDVPRDLEVVAATPTSLLISW RHPHFPTH
YYRITYGETGGNSPVQEFTVP LQPPA 173 M6 WT VSDVPRDLEVVAATPTSLLISW
RHPHFPTH YYRITYGETGGNSPVQEFTVP LQPPA 174 MWB6 WT
VSDVPRDLEVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPT 175
M5FL WT VSDVPRDLEVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPL
176 MWG10-f1 WT VSDVPRDLEVVAATPTSLLISW RHPHFPTR
YYRITYGETGGNSPVQEFTVP LQPPI 177 MWD08-f1; WT VSDVPRDLEVVAATPTSLLISW
RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPI N42G 178 M7 WT
VSDVPRDLEVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPTT 179 M1
WT VSDVPRDLEVVAATPTSLLISW RHPHFPTR YYRITYGETGGNSPVQEFTVP LQPTV 180
MWA07-f1 WT VSDVPRDLEVVAATPTSLLISW RPPHFPTR YYRITYGETGGNSPVQEFTVP
LQPTV 181 MWH11-f1 WT VSDVPRDLEVVAATPTSLLISW RHPHFPTR
YYRITYGETGGNSPVQEFTVP PQPPA 182 MWF09- WT VSDVPRDLEVVAATPTSLLISW
RHPHFPTR YYRITYGETGGNSPVQEFTVP PQPPA f1;F48S 183 MWG12-f1 WT
VSDVPRDLEVVAATPTSLLISW CHPHFPTR YYRITYGETGGNSPVQEFTVP LQPPI
TABLE-US-00018 SEQ Binding to Binding Kd Kd ID 1 nM to 1 nM KDR,
Flk, NO Framework 3 FG Loop Framework 4 KDR, % Flk-1, % nM nM 23
ATISGLKPGVDYTITGYAVT V E R N G R K L M T P ISINYRT 46 47 24
ATISGLKPGVDYTITGYAVT V E R N G R V L M T P ISINYRT 41 44 1.51 0.91
25 ATISGLKPGVDYTITGYAVT V E R N G R H L M T P ISINYRT 38 40 1.3
0.66 26 ATISGLKPGVDYTITGYAVT V E R N G R M L M T P ISINYRT 38 38 27
ATISGLKPGVDYTITGYAVT L E R N G R V L M T P ISINYRT 36 49 28
ATISGLKPGVDYTITGYAVT L E R N G R V L N T P ISINYRT 32 47 29
ATISGLKPGVDYTITGYAVT V E R N G R Q L M T P ISINYRT 42 33 30
ATISGLKPGVDYTITGYAVT V E R N G R T L F T P ISINYRT 27 44 31
ATISGLKPGVDYTITGYAVT L E R N G R E L M T P ISINYRT 41 28 1.58 1.3
32 ATISGLKPGVDYTITGYAVT L E R N G R L L N T P ISINYRT 33 40 33
ATISGLKPGVDYTITGYAVT H E R N G R V L M T P ISINYRT 32 40 34
ATISGLRPGVDYTITGYAVT E E R N G R V L F T P ISINYRT 31 40 35
ATISGLKPGVDYTITGYAVT V E R N G R Q L Y T P ISINYRT 34 38 36
ATISGLKPGVDYTITGYAVT V E R N G R A L M T P ISINYRT 36 30 37
ATISGLKPGVDYTITGYAVT V E R N G R N L M T P ISINYRT 35 30 38
ATISGLKPGVDYTITGYAVT L E R N G R V L I T P ISINYRT 30 34 39
ATISGLKPGVDYTITGYAVT V E R N G R V L N T P ISINYRT 26 41 40
ATISGLKPGVDYTITGYAVT V E R N G K V L M T P ISINYRT 39 27 41
ATISGLKPGVDYTITGYAVT V E R N G R T L M M P ISINYRT 38 23 42
ATISGLKPGVDYTITGYAVT M E R N G R E L M T P ISINYRT 33 27 43
ATISGLKPGVDYTITGYAVT E E R N G R T L R T P ISINYRT 24 34 2.37 1.4
44 ATISGLKPGVDYTITGYAVT V E R N G K T L M T P ISINYRT 32 30 45
ATISGLKPGVDYTITGYAVT L E R N D R V L L T P ISINYRT 31 30 46
ATISGLKPGVDYTITGYAVT L E R N G R K L M T P ISINYRT 30 29 47
ATISGLKPGVDYTITGYAVT V E P N G R V L N T P ISINYRT 32 23 48
ATISGLKPGVDYTITGYAVT V E R N D R V L F T P ISINYRT 24 29 49
ATISGLKPGVDYTITGYAVT V E R N G R E L K T P ISINYRT 29 21 50
ATISGLKPGVDYTITGYAVT V E R N G R E L R T P ISINYRT 29 21 51
ATISGLKPGVDYTITGYAVT Q E R N G R E L M T P ISINYRT 27 24 52
ATISGLKPGVDYTITGYAVT V E R N G R E L M T P ISINYRT 27 20 1.66 2.05
53 ATISGLKPGVDYTITGYAVT V E R N G R V L S V P ISINYRT 24 20 54
ATISGLKPGVDYTITGYAVT V E R D G R T L R T P ISINYRT 31 18 55
ATISGLKPGVDYTITGYAVT V E R N G R E L N T P ISINYRT 17 29 1.2 0.53
56 ATISGLKPGVDYTITGYAVT V E R N G R V L I V P ISINYRT 19 21 57
ATISGLKPGVDYTITGYAVT L E R N G R E L M V P ISINYRT 19 21 1.63 2.1
58 ATISGLKPGVDYTITGYAVT D G R N D R K L M V P ISINYRT 37 14 0.96
5.4 59 ATISGLKPGVDYTITGYAVT V E H N G R T S F T P ISINYRT 33 13 60
ATISGLKPGVDYTITGYAVT V E R D G R K L Y T P ISINYRT 27 15 61
ATISGLKPGVDYTITGYAVT L E R N G R E L N T P ISINYRT 15 23 62
ATISGLKPGVDYTITGYAVT D G W N G R L L S I P ISINYRT 36 7 0.35 7.1 63
ATISGLKPGVDYTITGYAVT D G Q N G R L L N V P ISINYRT 26 10 0.4 3.2 64
ATISGLKPGVDYTITGYAVT I E K N G R H L N I P ISINYRT 21 12 65
ATISGLKPGVDYTITGYAVT D G W N G K M L S V P ISINYRT 33 7 66
ATISGLKPGVDYTITGYAVT D G Y N D R L L F I P ISINYRT 46 2 67
ATISGLKPGVDYTITGYAVT D G P N D R L L N I P ISINYRT 18 2 68
ATISGLKPGVDYTITGYAVT D G P N N R E L I V P ISINYRT 18 2 69
ATISGLKPGVDYTITGYAVT D G L N G K Y L F V P ISINYRT 38 4 70
ATISGLKPGVDYTITGYAVT E G W N D R E L F V P ISINYRT 31 4 71
ATISGLKPGVDYTITGYAVT F G W N G R E L L T P ISINYRT 34 4 72
ATISGLKPGVDYTITGYAVT F G W N D R E L L I P ISINYRT 50 0 73
ATISGLKPGVDYTITGYAVT L E W N N R V L M T P ISINYRT 26 6 74
ATISGLKPGVDYTITGYAVT V E W N G R V L M T P ISINYRT 40 10 75
ATISGLKPGVDYTITGYAVT N E R N G R E L M T P ISINYRT 19 12 76
ATISGLKPGVDYTITGYAVT L E R N G K E L M T P ISINYRT 23 11 77
ATISGLKPGVDYTITGYAVT V E R N G R E L L T P ISINYRT 18 10 78
ATISGLKPGVDYTITGYAVT V E R N G R E L K T P ISINYRT 29 21 79
ATISGLKPGVDYTITGYAVT Q E R N G R E L R T P ISINYRT 28 13 80
ATISGLKPGVDYTITGYAVT V E R N G R E L L W P ISINYRT 40 16 81
ATISGLKPGVDYTITGYAVT L E R N G R E L M I P ISINYRT 31 17 82
ATISGLKPGVDYTITGYAVT V E R N G L V L M T P ISINYRT 33 7 83
ATISGLKPGVDYTITGYAVT V E R N G R V L I I P ISINYRT 24 17 84
ATISGLKPGVDYTITGYAVT V E R N G H K L F T P ISINYRT 24 3 85
ATISGLKPGVDYTITGYAVT V E R N E R V L M T P ISINYRT 26 20 86
ATISGLKPGVDYTITGYAVT F G P N D R E L L T P ISINYRT 32 1 87
ATISGLKPGVDYTITGYAVT M G P N D R E L L T P ISINYRT 37 1 88
ATISGLKPGVDYTITGYAVT M G K N D R E L L T P ISINYRT 32 1 89
ATISGLKPGVDYTITGYAVT V H W N G R E L M T P ISINYRT 36 7 90
ATISGLKPGVDYTITGYAVT E E W N G R V L M T P ISINYRT 51 10 91
ATISGLKPGVDYTITGYAVT V E R N G H T L M T P ISINYRT 37 9 92
ATISGLKPGVDYTITGYAVT V E E N G R Q L M T P ISINYRT 35 0 93
ATISGLKPGVDYTITGYAVT L E R N G Q V L F T P ISINYRT 33 11 94
ATISGLKPGVDYTITGYAVT V E R N G Q V L Y T P ISINYRT 43 11 95
ATISGLKPGVDYTITGYAVT W G Y K D H E L L I P ISINYRT 47 1 96
ATISGLKPGVDYTITGYAVT L G R N D R E L L T P ISINYRT 45 3 97
ATISGLKPGVDYTITGYAVT D G P N D R L L N I P ISINYRT 53 10 98
ATISGLKPGVDYTITGYAVT F A R D G H E I L T P ISINYRT 36 1 99
ATISGLKPGVDYTITGYAVT L E Q N G R E L M T P ISINYRT 38 1 100
ATISGLKPGVDYTITGYAVT V E E N G R V L N T P ISINYRT 32 10 101
ATISGLKPGVDYTITGYAVT L E P N G R Y L M V P ISINYRT 52 2 102
ATISGLKPGVDYTITGYAVT E G R N G R E L F I P ISINYRT 53 3 103
ATISGLKPGVDYTITGYAVT S G R N D R E L L V P ISINYRT 18 2 104
ATISGLRPGVDYTITGYAVT V E R D G R E L N I P ISINYRT 12 8 105
ATISGLKPGVDYTITGYAVT V E Q N G R V L M T P ISTNYRT 37 2 106
ATISGLKPGVDYTITGYAVT V E H N G R V L N I P ISINYRT 30 7 107
ATISGLKPGVDYTITGYAVT M A P N G R E L L T P ISINYRT 29 1 108
ATISGLKPGVDYTITGYAVT V E Q N G R V L N T P ISINYRT 20 8 109
ATISGLKPGVDYTITGYAVT D G R N G H E L M T P ISINYRT 17 1 110
ATISGLKPGVDYTITGYAVT E G R N G R E L M V P ISINYRT 22 2 111
ATISGLKPGVDYTITGYAVT L E R N N R E L L T P ISiNYRT 25 9 112
ATISGLKPGVDYTITGYAVT M E R S G R E L M T P ISINYRT 28 10 113
ATISGLKPGVDYTITGYAVT R A L L S I E L F T P ISINYRT 114
ATISGLKPGVDYTITGYAVT F A R K G T E L F T P ISINYRT 115
ATISGLKPGVDYTITGYAVT L E R C G R E L F T P ISINYRT 116
ATISGLKPGVDYTITGYAVT R E R N G R E L F T P ISINYRT 117
ATISGLKPGVDYTITGYAVT K E R N G R E L F T P ISINYRT 118
ATISGLKPGVDYTITGYAVT C E R N G R E L F T P ISINYRT 119
ATISGLKPGVDYTITGYAVT L E R T G R E L F T P ISTNYRT 120
ATISGLKPGVDYTITGYAVT W E R T G K E L F T P ISINYRT 121
ATISGLKPGVDYTITGYAVT I E R T C R E L F T P ISINYRT 122
ATISGLKPGVDYTITGYAVT G G M I V R E L F T P ISINYRT 123
ATISGLKPGVDYTITGYAVT F G R S S R E L F T P ISINYRT 124
ATISGLKPGVDYTITGYAVT R H K S R G E L F T P ISINYRT 125
ATISGLKPGVDYTITGYAVT R H R D K R E L F T P ISINYRT 126
ATISGLKPGVDYTITGYAVT Y H R G R G E L F T P ISINYRT 127
ATISGLKPGVDYTITGYAVT R H R G C R E L F T P ISINYRT 128
ATISGLKPGVDYTITGYAVT S H R L R K E L F T P ISINYRT 129
ATISGLKPGVDYTITGYAVT M H R Q R G E L F T P ISINYRT 130
ATISGLKPGVDYTITGYAVT F H R R R G E L F T P ISINYRT 131
ATISGLKPGVDYTITGYAVT F H R R R G E L F T P ISINYRT 132
ATISGLKPGVDYTITGYAVT S H R R R N E L F T P ISTNYRT 133
ATISGLKPGVDYTITGYAVT L H R R V R E L F T P ISTNYRT 134
ATISGLKPGVDYTITGYAVT R H R R R G E L F T P ISINYRT 135
ATISGLKPGVDYTITGYAVT W H R S R K E L F T P ISINYRT 136
ATISGLKPGVDYTITGYAVT R H R S R G E L F T P ISINYRT 137
ATISGLKPGVDYTITGYAVT V H R T G R E L F T P ISINYRT 138
ATISGLKPGVDYTITGYAVT W H R V R G E L F T P ISINYRT 139
ATISGLKPGVDYTITGYAVT W H R V R G E L F T P ISINYRT 140
ATISGLKPGVDYTITGYAVT W H R W R G E L F T P ISTNYRT 141
ATISGLKPGVDYTITGYAVT W K R S G G E L F T P ISINYRT 142
ATISGLKPGVDYTITGYAVT R L X N X V E L F T P ISINYRT 143
ATISGLKPGVDYTITGYAVT W R T P H A E L F T P ISINYRT 144
ATISGLKPGVDYTITGYAVT L S P H S V E L F T P ISINYRT
145 ATISGLKPGVDYTITGYAVT V S R Q K A E L F T P ISINYRT 146
ATISGLKPGVDYTITGYAVT S S Y S K L E L F T P ISINYRT 147
ATISGLKPGVDYTITGYAVT L T D R G S E L F T P ISINYRT 148
ATISGLKPGVDYTITGYAVT G T R T R S E L F T P ISINYRT 149
ATISGLKPGVDYTITGYAVT P V A G C S E L F T P ISINYRT 150
ATISGLKPGVDYTITGYAVT W W Q T P R E L F T P ISINYRT 151
ATISGLKPGVDYTITGYAVT W W Q T P R E L F T P ISINYRT 152
ATISGLKPGVDYTITGYAVT W E R N G R E L F T P ISINYRT 153
ATISGLKPGVDYTITGYAVT W E W N G R E L F T P ISINYRT 154
ATISGLKPGVDYTITGYAVT K E R N G R E L F T P ISINYRT 155
ATISGLKPGVDYTITGYAVT G A L N T S E L F T P ISINYRT 156
ATISGLKPGVDYTITGYAVT F G R E R R E L F T P ISINYRT 157
ATISGLKPGVDYTITGYAVT S G R V S F E L F T P ISTNYRT 158
ATISGLKPGVDYTITGYAVT F H R R R G E L F T P ISINYRT 159
ATISGLKPGVDYTITGYAVT L I R M N T E L F T P ISINYRT 160
ATISGLKPGVDYTITGYAVT C L H L I T E L F T P ISINYRT 161
ATISGLKPGVDYTITGYAVT V L K L T L E L F T P ISINYRT 162
ATISGLKPGVDYTITGYAVT V L K L T L E L F T P ISINYRT 163
ATISGLKPGVDYTITGYAVT V L K L T L E L F T P ISINYRT 164
ATISGLKPGVDYTITGYAVT A L M A S G E L F T P ISINYRT 165
ATISGLKPGVDYTITGYAVT S M K N R L E L F T P ISINYRT 166
ATISGLKPGVDYTITGYAVT L R C L I P E L F T P ISINYRT 167
ATISGLKPGVDYTITGYAVT V S R Q K A E L F T P ISINYRT 168
ATISGLKPGVDYTITGYAVT W S R T G R E L F T P ISINYRT 169
ATISGLKPGVDYTITGYAVT V W R T G R E L F T P ISTNYRT 170
ATISGLKPGVDYTITGYAVT T E R T G R E L F T P ISINYRT 171
ATISGLKPGVDYTITGYAVT K E R S G R E L F T P ISINYRT 172
ATISGLKPGVDYTITGYAVT L E R N D R E L F T P ISINYRT 173
ATISGLKPGVDYTITGYAVT L E R D G R E L F T P ISINYRT 174
ATISGLKPGVDYTITGYAVT Q G R H K R E L F T P ISINYRT 175
ATISGLKPGVDYTITGYAVT K E R N G R E L F T P ISINYRT 176
ATISGLKPGVDYTITGYAVT M A Q N D H E L I T P ISINYRT 177
ATISGLKPGVDYTITGYAVT M A Q N D H E L I T P ISINYRT 178
ATISGLKPGVDYTITGYAVT W E R N G R E L F T P ISINYRT 179
ATISGLKPGVDYTITGYAVT L E R N D R E L F T P ISINYRT 180
ATISGLKPGVDYTITGYAVT L E R N D R E L F T P ISINYRT 181
ATISGLKPGVDYTITGYAVT K E R S G R E L F T P ISINYRT 182
ATISGLKPGVDYTITGYAVT L E R N D R E L F T P ISINYRT 183
ATISGLKPGVDYTITGYAVT M A Q N D H E L I T P ISINYRT
TABLE-US-00019 TABLE 3 KDR binders Binding to Kd SEQ ID 1 nM KDR,
NO Clone Name N-terminus BC Loop DE Loop FG Loop KDR, % nM 6 K1 Del
1-8 RHPHFPTR LQPPT M G L Y G H E L L T P 48 0.55 7 K2 Del 1-8
RHPHFPTR LQPPT D G E N G Q F L L V P 48 1.19 8 K5 Del 1-8 RHPHFPTR
LQPPT M G P N D N E L L T P 47 1.54 9 K3 Del 1-8 RHPHFPTR LQPPT A G
W D D H E L F I P 45 1.15 311 3'E9 PR4 Del 1-8 RHPHFPTR LQPPT V E Q
D G H V L Y I P 44 312 2'Del E6 PR4 Del 1-8 RHPHFPTR LQPPT M G K N
G H E L L T P 43 313 3'D3 PR4 Del 1-8 RHPHFPTR LQPPT P G P G D R E
L I T P 42 314 2'Del F8 PR4 Del 1-8 RHPHFPTR LQPPT A G P G A H E L
L T P 42 315 4'B3 PR4 Del 1-8 RHPHFPTR LQPPT M A Q N N R E L L T P
42 316 3'E3 PR4 Del 1-8 RHPHFPTR LQPPT M A Q Y G R E L L T P 41 10
K7 Del 1-8 RHPHFPTR LQPPT S G H N D H M L M I P 40 2.2 317 3'H11
PR4 Del 1-8 RHPHFPTR LQPPT L A H N G N E L L T P 39 318 3'B4 PR4
Del 1-8 RHPHFPTR LQPPT V A W N G H E L M T P 38 11 K4 Del 1-8
RHPHFPTR LQPPT A G Y N D Q I L M T P 38 1.95 319 2'Del F7 PR4 Del
1-8 RHPHFPTR LQPPT L G L R D R E L F V P 38 320 2'Del D3 PR4 Del
1-8 RHPHFPTR LQPPT S G L N D R V L F I P 38 321 3'C6 PR4 Del 1-8
RHPHFPTR LQPPT M G P N D R E L L T P 37 322 3'F3 PR4 Del 1-8
RHPHFPTR LQPPT L G H N D R E L L T P 37 323 3'H3 PR4 Del 1-8
RHPHFPTR LQPPT L G L N D R E L M T P 36 324 1'Del G10 PR4 Del 1-8
RHPHFPTR LQPPT M A Q N G H K L M T P 36 12 K9 Del 1-8 RHPHFPTR
LQPPT F G L Y G K E L L I P 35 1.8 325 2'DelE4 PR4 Del 1-8 RHPHFPTR
LQPPT V H W N G H E L M T P 34 326 2'Del C6 PR4 Del 1-8 RHPHFPTR
LQPPT M G F M A H E L M V P 34 327 2'Del C11 PR4 Del 1-8 RHPHFPTR
LQPPT A G L N E H E L L I P 34 328 2'Del D10 PR4 Del 1-8 RHPHFPTR
LQPPT L A D N A R E L L T P 34 329 2'Del H5 PR4 Del 1-8 RHPHFPTR
LQPPT L G K D V R E L L T P 34 330 3'A7 PR4 Del 1-8 RHPHFPTR LQPPT
L S D S G H A L F T P 34 331 2'Del E3 PR4 Del 1-8 RHPHFPTR LQPPT L
G P Y E H E L L T P 33 13 K10 Del 1-8 RHPHFPTR LQPPT T G P N D R L
L F V P 33 0.57 332 2'Del B5 PR4 Del 1-8 RHPHFPTR LQPPT A G R H D H
E L I I P 33 333 3'C12 PR4 Del 1-8 RHPHFPTR LQPPT I G P N N H E L L
T P 33 334 2'Del G9 PR4 Del 1-8 RHPHFPTR LQPPT V E Q N G R E L I I
P 33 335 2'Del C1 PR4 Del 1-8 RHPHFPTR LQPPT A G L D E H E L L I P
32 336 3'E1 PR4 Del 1-8 RHPHFPTR LQPPT V A P N G H E L F T P 32 337
3'C3 PR4 Del 1-8 RHPHFPTR LQPPT M A Q N G H A L F T P 32 338
2'DelB7 PR4 Del 1-8 RHPHFPTR LQPPT V G Y N N R E L L T P 32 339
3'F1 PR4 Del 1-8 RHPHFPTR LQPPT V A Q D G H F L Y T P 31 340 2'Del
B4 PR4 Del 1-8 RHPHFPTR LQPPT S G H N G H E V M T P 31 341 3'G3 PR4
Del 1-8 RHPHFPTR LQPPT F D Q S D H E L L T P 31 342 2'DelH4 PR4 Del
1-8 RHPHFPTR LQPPT V G P N E R M L M T P 30 343 3'D9 PR4 Del 1-8
RHPHFPTR LQPPT G Y Y N D R E L L T P 30 344 3'G10 PR4 Del 1-8
RHPHFPTR LQPPT L T H N D H E L L T P 30 345 3'B2 PR4 Del 1-8
RHPHFPTR LQPPT V G R N D R E L L T P 29 346 2'DelC3 PR4 Del 1-8
RHPHFPTR LQPPT W A Q N G R E L L T P 29 347 3'F2 PR4 Del 1-8
RHPHFPTR LQPPT L G K N D H E L L T P 29 348 4'C9 PR4 Del 1-8
RHPHFPTR LQPPT L G P N D H E L M T P 29 349 2'Del B2 PR4 Del 1-8
RHPHFPTR LQPPT T G W N G N E L F T P 29 14 K12 Del 1-8 RHPHFPTR
LQPPT D V Y N D H E I K T P 29 0.62 350 4'H7 PR4 Del 1-8 RHPHFPTR
LQPPT L A H N D H E L L T P 29 351 2'Del D1 PR4 Del 1-8 RHPHFPTR
LQPPT L E Q N D R V L L T P 28 352 2'Del H6 PR4 Del 1-8 RHPHFPTR
LQPPT T G H H D H E L I I P 28 353 3'B12 PR4 Del 1-8 RHPHFPTR LQPPT
V A H E N R E L L T P 28 354 4'C5 PR4 Del 1-8 RHPHFPTR LQPPT L G L
N D H E L I T P 27 15 K6 Del 1-8 RHPHFPTR LQPPT D G K D G R V L L T
P 27 0.93 355 3'D8 PR4 Del 1-8 RHPHFPTR LQPPT A G P N D H Q L F T P
27 356 3'C5 PR4 Del 1-8 RHPHFPTR LQPPT D A M Y G R E L M T P 27 357
3'A8 PR4 Del 1-8 RHPHFPTR LQPPT V A W D D H E L L T P 27 358 2'Del
F11 PR4 Del 1-8 RHPHFPTR LQPPT M G Q N D K E L I T P 27 359 4'D8
PR4 Del 1-8 RHPHFPTR LQPPT L A Q N G H E L Y T P 26 360 2'Del C5
PR4 Del 1-8 RHPHFPTR LQPPT P G H N D H E L M V P 26 16 K15 Del 1-8
RHPHFPTR LQPPT E V H H D R E I K T P 25 0.35 361 3'B1 PR4 Del 1-8
RHPHFPTR LQPPT E A R N G R E L L T P 25 362 3'A9 PR4 Del 1-8
RHPHFPTR LQPPT L A H N D R E L L T P 25 363 4'B11 PR4 Del 1-8
RHPHFPTR LQPPT M A H N D H E L L T P 25 17 K11 Del 1-8 RHPHFPTR
LQPPT Q A P N D R V L Y T P 24 1.16 364 3D12 PR3 Del 1-8 RHPHFPTR
LQPPT L G Q N D R Q L L V P 24 365 2'Del H12 PR4 Del 1-8 RHPHFPTR
LQPPT A G G N G H E L L T P 24 366 3'H9 PR4 Del 1-8 RHPHFPTR LQPPT
H G P Y D Q V L L T P 24 367 3'F6 PR4 Del 1-8 RHPHFPTR LQPPT I E Q
S G L Q L M T P 24 368 1'DelE6 PR4 Del 1-8 RHPHFPTR LQPPT L A Q N D
R E L L T P 24 369 3'E5 PR4 Del 1-8 RHPHFPTR LQPPT V A W D G R E L
F T P 23 370 3A3 PR3 Del 1-8 RHPHFPTR LQPPT L A Y N G R E I I T P
23 371 3A2 PR3 Del 1-8 RHPHFPTR LQPPT W S Q N N R E L F T P 23 372
3'B11 PR4 Del 1-8 RHPHFPTR LQPPT E T W N D H E I R T P 23 373
1'DelA2 PR4 Del 1-8 RHPHFPTR LQPPT V A Q N G H Q L F T P 23 374
2'D6-PR4 WT RHPHFPTR LQPPT V T H N G H P L M T P 22 375 3'H1 PR4
Del 1-8 RHPHFPTR LQPPT F A Q N D H Q L F T P 22 376 2'Del G11 PR4
Del 1-8 RHPHFPTR LQPPT G G Q M N R V L M T P 22 377 2'Del F5 PR4
Del 1-8 RHPHFPTR LQPPT L V H N D R E L L T P 22 378 1'DelE7 PR4 Del
1-8 RHPHFPTR LQPPT V A Q N G H E L F T P 22 379 2'E4-PR4 WT
RHPHFPTR LQPPT V H W N G H E L M T P 22 380 2'Del F6 PR4 Del 1-8
RHPHFPTR LQPPT L G W N D H E L Y I P 22 381 3'E10 PR4 Del 1-8
RHPHFPTR LQPPT A G H K D Q E L L T P 21 382 4'A9 PR4 Del 1-8
RHPHFPTR LQPPT L A Q N N H E L L T P 21 383 4'G12 PR4 Del 1-8
RHPHFPTR LQPPT V A W N D H E I Y T P 21 384 3'B10 PR4 Del 1-8
RHPHFPTR LQPPT L A Q T G R E L L T P 21 385 2'DelH9 PR4 Del 1-8
RHPHFPTR LQPPT V G W S G H E L F V P 20 386 3'H8 PR4 Del 1-8
RHPHFPTR LQPPT V G H N D R E L I T P 20 387 2'DelA5 PR4 Del 1-8
RHPHFPTR LQPPT W N Q N G Q E L F T P 20 388 3B5 PR3 Del 1-8
RHPHFPTR LQPPT F G Q N G H A L L T P 20 389 3C7 PR3 Del 1-8
RHPHFPTR LQPPT R G L N D G E L L T P 20 390 3G2 PR3 Del 1-8
RHPHFPTR LQPPT F G P S D H V L L T P 20 18 K14 Del 1-8 RHPHFPTR
LQPPT R E E N D H E L L I P 20 0.57 391 4'B12 PR4 Del 1-8 RHPHFPTR
LQPPT L A Q N N H E L L T P 20 392 4'B8 PR4 Del 1-8 RHPHFPTR LQPPT
V A Q N D H K L F I P 20 393 2'Del F1 PR4 Del 1-8 RHPHFPTR LQPPT R
D Q Y E H E L L T P 20 394 3'G1 PR4 Del 1-8 RHPHFPTR LQPPT L A L N
G H E L F T P 19 395 3'D2 PR4 Del 1-8 RHPHFPTR LQPPT V E S N G H A
L F V P 19 396 2'DelG5 PR4 Del 1-8 RHPHFPTR LQPPT V G Q N N H E L L
T P 19 397 2'DelC7 PR4 Del 1-8 RHPHFPTR LQPPT W D Q N G H V L L T P
19 398 2'Del E5 PR4 Del 1-8 RHPHFPTR LQPPT E G L N D H E L I I P 19
399 3'C8 PR4 Del 1-8 RHPHFPTR LQPPT E G L N D H E L M I P 19 400
3'G7 PR4 Del 1-8 RHPHFPTR LQPPT E G Q N D Q L L F I P 19 401 3'A6
PR4 Del 1-8 RHPHFPTR LQPPT L A Q N G H E L L T P 19 402 4'G4 PR4
Del 1-8 RHPHFPTR LQPPT V A Q N D R E L L T P 19 403 4'H5 PR4 Del
1-8 RHPHFPTR LQPPT L A Q N G H E L F T P 18 404 1'DelH12 PR4 Del
1-8 RHPHFPTR LQPPT V A Q N E R E L F T P 18 19 K8 Del 1-8 RHPHFPTR
LQPPT V T H N G H P L M T P 18 3.3 405 2'Del D5 PR4 Del 1-8
RHPHFPTR LQPPT V A W N D H M L M T P 18 406 3'F9 PR4 Del 1-8
RHPHFPTR LQPPT L G P N D R E L M T P 18 407 2'H4-PR4 WT RHPHFPTR
LQPPT V G P N E R M L M T P 17 408 4'H12 PR4 Del 1-8 RHPHFPTR LQPPT
V A H N D H E L L T P 17 409 1'DelD2 PR4 Del 1-8 RHPHFPTR LQPPT V A
K N D H E L L T P 17 410 4'E7 PR4 Del 1-8 RHPHFPTR LQPPT W A Q N D
H E L L T P 17 411 1'DelH10 PR4 Del 1-8 RHPHFPTR LQPPT F A Q N D H
E L L T P 17 20 K13 Del 1-8 RHPHFPTR LQPPT L A L K G H E L L T P 17
0.58 412 3C3 PR3 Del 1-8 RHPHFPTR LQPPT M E Q N G H E L F T P 17
413 2'Del B3 PR4 Del 1-8 RHPHFPTR LQPPT D A P N G R E L M V P 17
414 2'Del A2 PR4 Del 1-8 RHPHFPTR LQPPT G G R N G H T L L T P 17
415 3'F12 PR4 Del 1-8 RHPHFPTR LQPPT L S Q T D H E L L T P 17 416
3B4 PR3 Del 1-8 RHPHFPTR LQPPT V G Q N E H E L L T P 17 417 3F8 PR3
Del 1-8 RHPHFPTR LQPPT V A Q N G H E L K T P 17 418 1'DelH5 PR4 Del
1-8 RHPHFPTR LQPPT V A Q N D R E L F T P 17 419 1'DelD5 PR4 Del 1-8
RHPHFPTR LQPPT V G Q N H H E L F T P 17 420 3'E11 PR4 Del 1-8
RHPHFPTR LQPPT V G P H D R E L L T P 17 421 2'C6-PR4 WT RHPHFPTR
LQPPT M G F M A H E L M V P 16 422 4C9 PR3 Del 1-8 RHPHFPTR LQPPT L
A Q N D H E L L T P 16 423 3C9 PR3 Del 1-8 RHPHFPTR LQPPT L V R N D
H E L L T P 16 424 3F10 PR3 Del 1-8 RHPHFPTR LQPPT L A Q D D H E L
L T P 16 425 2'Del A11 PR4 Del 1-8 RHPHFPTR LQPPT E D I R V L W L N
T T 16 426 1'DelD1 PR4 Del 1-8 RHPHFPTR LQPPT V T Q N D H E L L T P
16 427 1'DelE2 PR4 Del 1-8 RHPHFPTR LQPPT V G Q N D H E L L T P 16
428 1'DelF3 PR4 Del 1-8 RHPHFPTR LQPPT M A Q N D H K L F T P 16 429
4'A5 PR4 Del 1-8 RHPHFPTR LQPPT L A Q N D H E L L T P 16 430
1'DelB8 PR4 Del 1-8 RHPHFPTR LQPPT M A Q N D H E L L T P 16 431
4'B7 PR4 Del 1-8 RHPHFPTR LQPPT V A Q N N H E L L T P 16 432 4F4
PR3 Del 1-8 RHPHFPTR LQPPT L A Q N D R E L I T P 15 433 4B11 PR3
Del 1-8 RHPHFPTR LQPPT V G Q N N H E L I T P 15 434 3'G2 PR4 Del
1-8 RHPHFPTR LQPPT L A Q N G H E L I T P 15 435 2'Del C8 PR4 Del
1-8 RHPHFPTR LQPPT T A H N G H E L L T P 15 436 3'B8 PR4 Del 1-8
RHPHFPTR LQPPT L G Y H D H A L F T P 14 437 3'H10 PR4 Del 1-8
RHPHFPTR LQPPT W A W N D H E L M T P 14 438 1'DelA1 PR4 Del 1-8
RHPHFPTR LQPPT V A Q N D H E L L T P 14 439 4'D6 PR4 Del 1-8
RHPHFPTR LQPPT M A Q N D H E L M T P 14 440 4F9 PR3 Del 1-8
RHPHFPTR LQPPT M A Q N D H E L L T P 14 441 4H5 PR3 Del 1-8
RHPHFPTR LQPPT V A Q N G H E L I T P 14 442 2D12 PR3 WT RHPHFPTR
LQPPT E G W I D H E I M I P 14 443 3'F7 PR4 Del 1-8 RHPHFPTR LQPPT
E G Q N G S E L I V P 14 444 4C11 PR3 Del 1-8 RHPHFPTR LQPPT M A Q
N D R E L I T P 14 445 4B6 PR3 Del 1-8 RHPHFPTR LQPPT V G Q N D H E
L F T P 14 446 1'DelE12 PR4 Del 1-8 RHPHFPTR LQPPT V A Q S D H E L
F T P 13 447 1'DelC2 PR4 Del 1-8 RHPHFPTR LQPPT V D R N D H E L F T
P 13 448 1'DelA9 PR4 Del 1-8 RHPHFPTR LQPPT L A Q N D H E L M T P
13 449 1'DelA4 PR4 Del 1-8 RHPHFPTR LQPPT V A Q N D H E L F T P 13
450 3G5 PR3 Del 1-8 RHPHFPTR LQPPT L G E N D R K L I T P 13 451
4A12 PR3 Del 1-8 RHPHFPTR LQPPT V A Q N D H E L L T P 13 452 2'Del
E12 PR4 Del 1-8 RHPHFPTR LQPPT E G P N G H E L I T P 13 453 3G1 PR3
Del 1-8 RHPHFPTR LQPPT M A Q N V R E L L T P 13 454 4F12 PR3 Del
1-8 RHPHFPTR LQPPT V T Q N G H E L I T P 13 455 4B7 PR3 Del 1-8
RHPHFPTR LQPPT V T Q N D H E L F T P 13 456 4'G8 PR4 Del 1-8
RHPHFPTR LQPPT V A Q N G H E L L T P 13 457 3'E8 PR4 Del 1-8
RHPHFPTR LQPPT V A Q N D R Q L F T P 12 458 3'E4 PR4 Del 1-8
RHPHFPTR LQPPT V G P N D R E L I V P 12 459 1'DelC6 PR4 Del 1-8
RHPHFPTR LQPPT V A Q N E H E L L T P 12 460 1'DelD3 PR4 Del 1-8
RHPHFPTR LQPPT L A Q N N H E L I T P 12 461 3A8 PR3 Del 1-8
RHPHFPTR LQPPT E A H H G H E L M I P 12 462 3C5 PR3 Del 1-8
RHPHFPTR LQPPT G D H N D R E L M T P 12 463 2'G11-PR4 WT RHPHFPTR
LQPPT G G Q M N R V L M T P 12 464 3'D4 PR4 Del 1-8 RHPHFPTR LQPPT
L A H N D R E L I T P 12 465 3E6 PR3 Del 1-8 RHPHFPTR LQPPT V P Q N
G H E L I T M 12 466 1'DelA11 PR4 Del 1-8 RHPHFPTR LQPPT L A Q N D
H E L F T P 12 467 4'D12 PR4 Del 1-8 RHPHFPTR LQPPT V D Q N D H E L
F T P 12 468 2'D5-PR4 WT RHPHFPTR LQPPT V A W N D H M L M T P 11
469 2'A1-PR4 WT RHPHFPTR LQPPT S G H N D H M L M I P 11 470
1'DelG11 PR4 Del 1-8 RHPHFPTR LQPPT L A Q N G H V L I T P 11 471
2'DelB10 PR4 Del 1-8 RHPHFPTR LQPPT V T H N D H E L L T P 11 472
2'DelB11 PR4 Del 1-8 RHPHFPTR LQPPT V G Q N D H E L M T P 11 473
1'DelC5 PR4 Del 1-8 RHPHFPTR LQPPT L A Q N D H E I M T P 11 474
4'B6 PR4 Del 1-8 RHPHFPTR LQPPT L A Q N D H E L I T P 11 475 3H9
PR3 Del 1-8 RHPHFPTR LQPPT V S Q Q N H E L L T P 11 476 4E10 PR3
Del 1-8 RHPHFPTR LQPPT V A Q N D H E L M T P 11 477 3F5 PR3 Del 1-8
RHPHFPTR LQPPT V A Y N E H E L Y T P 11 478 4A9 PR3 Del 1-8
RHPHFPTR LQPPT V A Q H D H E L L T P 11 479 1'DelH7 PR4 Del 1-8
RHPHFPTR LQPPT V G Q N D Q E L L T P 11 480 1'DelB10 PR4 Del 1-8
RHPHFPTR LQPPT V A R N D H E L M T P 11 481 2'DelB9 PR4 Del 1-8
RHPHFPTR LQPPT V G P T D H E L L T P 11 482 3F11 PR3 Del 1-8
RHPHFPTR LQPPT V G L T D H V L L T P 10 483 4C4 PR3 Del 1-8
RHPHFPTR LQPPT V A Q D D H E L F T P 10 484 4B5 PR3 Del 1-8
RHPHFPTR LQPPT L A Q N D H E L F T P 10 485 3D4 PR3 Del 1-8
RHPHFPTR LQPPT V G W N D H E L I T P 10 486 4A4 PR3 Del 1-8
RHPHFPTR LQPPT V A Q N D H E L F T P 10 487 3D11 PR3 Del 1-8
RHPHFPTR LQPPT L G Q E N Q E L I T P 10 488 2H10 PR3 WT RHPHFPTR
LQPPT L A P S A R E L M T P 10 489 3G10 PR3 Del 1-8 RHPHFPTR LQPPT
V V H N G H E I L T P 10 490 3F4 PR3 Del 1-8 RHPHFPTR LQPPT M G Y E
D H E L I T P 10 491 2H12 PR3 WT RHPHFPTR LQPPT E G Y Q N H E L S V
P 10 492 4C2 PR3 Del 1-8 RHPHFPTR LQPPT V D Q N D H E L F T P 10
493 1'DelG9 PR4 Del 1-8 RHPHFPTR LQPPT V A Q S D H E L M T P 10 494
1'DelH9 PR4 Del 1-8 RHPHFPTR LQPPT V G Q N D H E L I T P 10 495
1'DelB3 PR4 Del 1-8 RHPHFPTR LQPPT V A Q N D H E L M T P 10 496
1'DelH1 PR4 Del 1-8 RHPHFPTR LQPPT V A Q N G H E L I T P 9 497 3'A3
PR4 Del 1-8 RHPHFPTR LQPPT R A Q N D H E L I T P 9 498 1'DelC4 PR4
Del 1-8 RHPHFPTR LQPPT V A Q S N H E L M T P 9 499 1'DelE11 PR4 Del
1-8 RHPHFPTR LQPPT V A Q N D R E L I T P 9 500 3F1 PR3 Del 1-8
RHPHFPTR LQPPT L T H N E Q Y L F T P 9 501 2G9 PR3 WT RHPHFPTR
LQPPT E I Y N D H E L M T P 9 502 3'D11 PR4 Del 1-8 RHPHFPTR LQPPT
M A Q N D H E L I T P 9 503 2'DelH2 PR4 Del 1-8 RHPHFPTR LQPPT V S
Q Y G H E L I T P 8 504 1'DelC10 PR4 Del 1-8 RHPHFPTR LQPPT V A K N
D H E L I T P 8 505 4D2 PR3 Del 1-8 RHPHFPTR LQPPT V A Q N N H E L
I T P 8 506 4A9 PR3 Del 1-8 RHPHFPTR LQPPT V A Q H D H E L L T P 8
507 2F3 PR3 WT RHPHFPTR LQPPT L S H Y G K E L R T P 8 508 4A2 PR3
Del 1-8 RHPHFPTR LQPPT V A Q N A H E L M T P 8 509 4G4 PR3 Del 1-8
RHPHFPTR LQPPT L G Q N D H E L L T P 8 510 1'DelB7 PR4 Del 1-8
RHPHFPTR LQPPT V A Q N D H E L K T P 8 511 3'D12 PR4 Del 1-8
RHPHFPTR LQPPT G E Q N D Y E L L V P 7 512 2'Del F12 PR4 Del 1-8
RHPHFPTR LQPPT L T Q H D H E L L T P 7 513 4E2 PR3 Del 1-8 RHPHFPTR
LQPPT M A Q N D H E L I T P 7 514 2C9 PR3 WT RHPHFPTR LQPPT E A P N
G R E L R T P 7 515 2'B9-PR4 WT RHPHFPTR LQPPT V G P T D H E L L T
P 7 516 1'DelH6 PR4 Del 1-8 RHPHFPTR LQPPT V G Q Y D H E L I T P 6
517 4A3 PR3 Del 1-8 RHPHFPTR LQPPT V A Q D E H E L I T P 6 518 2C12
PR3 WT RHPHFPTR LQPPT D A Q N V Q A P I A Q 6 519 2G12 PR3 WT
RHPHFPTR LQPPT S G Q N D H A L L I P 6 520 2'A11-PR4 WT RHPHFPTR
LQPPT E D I R V L W L N T T 6 521 2'C7-PR4 WT RHPHFPTR LQPPT W D Q
N G H V L L T P 5 522 2'F7-PR4 WT RHPHFPTR LQPPT L G L R D R E L F
V P 5 523 2C6 PR3 WT RHPHFPTR LQPPT V E P N G H K L A I P 5 524
3'E6 PR4 Del 1-8 RHPHFPTR LQPPT F G Q N G K E F R I P 5 525
2'B4-PR4 WT RHPHFPTR LQPPT S G H N G H E V M T P 4 526 2'F6-PR4 WT
RHPHFPTR LQPPT L G W N D H E L Y I P 4 527 2'H5-PR4 WT RHPHFPTR
LQPPT L G K D V R E L L T P 3 528 2F10 PR3 WT RHPHFPTR LQPPT L A L
F D H E L L T P 3 21 VR28 WT RHPHFPTR LQPPT V A Q N D H E L I T P 3
11 22 159 WT RHPHFPTR LQPPA M A Q S G H E L F T P
TABLE-US-00020 TABLE 4 Sequences of characterized VEGF-R2 binding
clones ##STR00001## ##STR00002## ##STR00003## ##STR00004##
##STR00005## ##STR00006## ##STR00007## ##STR00008## ##STR00009##
##STR00010## ##STR00011## ##STR00012## ##STR00013## ##STR00014##
##STR00015## ##STR00016## ##STR00017## ##STR00018## ##STR00019##
##STR00020## ##STR00021## ##STR00022## ##STR00023## ##STR00024##
##STR00025## ##STR00026## ##STR00027## ##STR00028##
TABLE-US-00021 TABLE 5 Affinities of the trinectin binders to
KDR-Fc determined in radioactive equilibrium binding assay Clone
KDR (Kd, nM) VR28 11.0 .+-. 0.5 K1 <0.6 .+-. 0.1 K6 <0.9 .+-.
0.1 K9 <1.8 .+-. 0.4 K10 <0.6 .+-. 0.1 K12 <0.6 .+-. 0.1
K13 <0.6 .+-. 0.1 K14 <0.6 .+-. 0.1 K15 <0.4 .+-. 0.1
TABLE-US-00022 TABLE 6 Affinities of the trinectin binders to KDR
and Flk-1 determined in radioactive equilibrium binding assay Clone
KDR (Kd, nM) Flk-1 (Kd, nM) VR28 11.0 .+-. 0.5 nd* E3 <1.0 .+-.
0.2 5.4 .+-. 1.5 E5 <0.4 .+-. 0.1 3.2 .+-. 0.3 E6 <0.4 .+-.
0.1 7.1 .+-. 1.1 E9 2.4 .+-. 0.3 <1.4 .+-. 0.1 E18 <1.2 .+-.
0.2 <0.5 .+-. 0.1 E19 <1.3 .+-. 0.2 <0.7 .+-. 0.1 E25
<1.6 .+-. 0.4 <1.3 .+-. 0.2 E26 <1.7 .+-. 0.4 2.0 .+-. 0.3
E28 <1.6 .+-. 0.4 2.1 .+-. 0.6 E29 <1.5 .+-. 0.4 <0.9 .+-.
0.2 nd* - binding is not detected at 100 nM of target
TABLE-US-00023 TABLE 7 Determination of ka, kd and Kd by BIAcore
assay Clone Target ka (1/M * s) .times. 10.sup.-4 kd(1/s) .times.
10.sup.+5 Kd (nM) E6 KDR 89 6.7 0.08 Flk-1 67 136.0 2.02 E18 KDR 26
12.1 0.46 Flk-1 60 19.5 0.33 E19 KDR 30 1.7 0.06 Flk-1 66 22.3 0.34
E25 KDR 25 5.2 0.21 Flk-1 50 37.8 0.76 E26 KDR 11 5.8 0.51 Flk-1 22
47.7 2.14 E29 KDR 36 7.0 0.19 Flk-1 79 28.8 0.37 M5FL KDR 10 9.2
0.89 Flk-1 28 58.2 2.10 VR28 KDR 3 34 13 159(Q8L) KDR 5 10 2
TABLE-US-00024 TABLE 8 Binding to KDR (CHO KDR) and Flk-1 (CHO
Flk-1) expressing cells CHO KDR CHO Flk-1 Clone (EC50, nM) (EC50,
nM) E18 4.2 .+-. 1.0 0.9 .+-. 0.4 E19 7.6 .+-. 1.7 5.3 .+-. 2.5 E26
2.6 .+-. 1.2 1.3 .+-. 0.7 E29 2.3 .+-. 1.0 0.6 .+-. 0.1 WT no
no
TABLE-US-00025 TABLE 9 Inhibition of VEGF-induced proliferation of
KDR (Ba/F3-KDR) and Flk-1 (Ba/F3-Flk) expressing cells Ba/F3-KDR
Ba/F3-Flk Clone (IC50, nM) (IC50, nM) E18 5.4 .+-. 1.2 2.4 .+-. 0.2
E19 12.3 .+-. 2.6 5.8 .+-. 1.0 E26 3.2 .+-. 0.5 5.3 .+-. 1.7 E29
10.0 .+-. 2.1 4.7 .+-. 1.2 M5FL 3.9 .+-. 1.1 5.1 .+-. 0.2 WT no no
Anti-KDR Ab 17.3 .+-. 7.7 ND Anti-Flk-1 Ab ND 15.0 .+-. 3.2
TABLE-US-00026 TABLE 10 Inhibition of VEGF-induced proliferation of
HUVEC cells Clone (IC50, nM) E18 12.8 .+-. 4.6 E19 11.8 .+-. 2.7
E26 14.0 .+-. 5.9 E29 8.4 .+-. 0.8 M5FL 8.5 .+-. 2.8 WT no
TABLE-US-00027 TABLE 11 hKDR Flk-1 k.sub.a k.sub.d k.sub.a k.sub.d
(1/Ms) .times. (1/s) .times. K.sub.D (1/Ms) .times. (1/s) .times.
K.sub.D 10.sup.-4 10.sup.5 (nM) 10.sup.-4 10.sup.5 (nM) M5FL 7.4
6.7 0.9 14.6 30 2.1 C100 M5FL 20K 0.9 5.4 5.9 2.4 55 22.8 PEG M5FL
40K 0.5 5.9 1.3 1.0 54 57.1 PEG
[0261] All references cited herein are hereby incorporated by
reference in their entirety
Sequence CWU 1
1
528111PRTArtificial Sequencebinding polypeptide 1Xaa Gly Xaa Asn
Xaa Xaa Glu Leu Xaa Thr Pro 1 5 10211PRTArtificial Sequencebinding
polypeptide 2Xaa Glu Arg Asn Gly Arg Xaa Leu Xaa Thr Pro 1 5
10310PRTArtificial Sequencebinding polypeptide 3Xaa Gly Xaa Asn Xaa
Arg Xaa Xaa Ile Pro 1 5 10410PRTArtificial Sequencebinding
polypeptide 4Xaa Gly Xaa Asn Xaa Arg Xaa Xaa Ile Pro 1 5
10594PRTHomo sapiens 5Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr Pro Thr 1 5 10 15Ser Leu Leu Ile Ser Trp Asp Ala Pro
Ala Val Thr Val Arg Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu Thr Gly
Gly Asn Ser Pro Val Gln Glu Phe 35 40 45Thr Val Pro Gly Ser Lys Ser
Thr Ala Thr Ile Ser Gly Leu Lys Pro 50 55 60Gly Val Asp Tyr Thr Ile
Thr Gly Tyr Ala Val Thr Gly Arg Gly Asp65 70 75 80Ser Pro Ala Ser
Ser Lys Pro Ile Ser Ile Asn Tyr Arg Thr 85 90686PRTArtificial
Sequencebinding polypeptide 6Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Met Gly Leu Tyr Gly His Glu Leu Leu Thr Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 85786PRTArtificial Sequencebinding polypeptide 7Glu
Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10
15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly
20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro
Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile
Thr Gly 50 55 60Tyr Ala Val Thr Asp Gly Glu Asn Gly Gln Phe Leu Leu
Val Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 85886PRTArtificial
Sequencebinding polypeptide 8Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Met Gly Pro Asn Asp Asn Glu Leu Leu Thr Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 85986PRTArtificial Sequencebinding polypeptide 9Glu
Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10
15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly
20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro
Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile
Thr Gly 50 55 60Tyr Ala Val Thr Ala Gly Trp Asp Asp His Glu Leu Phe
Ile Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 851086PRTArtificial
Sequencebinding polypeptide 10Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Ser Gly His Asn Asp His Met Leu Met Ile Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 851186PRTArtificial Sequencebinding polypeptide
11Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1
5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro
Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr
Ile Thr Gly 50 55 60Tyr Ala Val Thr Ala Gly Tyr Asn Asp Gln Ile Leu
Met Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
851286PRTArtificial Sequencebinding polypeptide 12Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Phe Gly Leu Tyr Gly Lys Glu Leu Leu Ile Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 851386PRTArtificial
Sequencebinding polypeptide 13Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Thr Gly Pro Asn Asp Arg Leu Leu Phe Val Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 851486PRTArtificial Sequencebinding polypeptide
14Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1
5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro
Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr
Ile Thr Gly 50 55 60Tyr Ala Val Thr Asp Val Tyr Asn Asp His Glu Ile
Lys Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
851586PRTArtificial Sequencebinding polypeptide 15Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Asp Gly Lys Asp Gly Arg Val Leu Leu Thr Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 851686PRTArtificial
Sequencebinding polypeptide 16Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Glu Val His His Asp Arg Glu Ile Lys Thr Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 851786PRTArtificial Sequencebinding polypeptide
17Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1
5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro
Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr
Ile Thr Gly 50 55 60Tyr Ala Val Thr Gln Ala Pro Asn Asp Arg Val Leu
Tyr Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
851886PRTArtificial Sequencebinding polypeptide 18Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Arg Glu Glu Asn Asp His Glu Leu Leu Ile Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 851986PRTArtificial
Sequencebinding polypeptide 19Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Val Thr His Asn Gly His Pro Leu Met Thr Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 852086PRTArtificial Sequencebinding polypeptide
20Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1
5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro
Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr
Ile Thr Gly 50 55 60Tyr Ala Val Thr Leu Ala Leu Lys Gly His Glu Leu
Leu Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
852194PRTArtificial Sequencebinding polypeptide 21Val Ser Asp Val
Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr 1 5 10 15Ser Leu
Leu Ile Ser Trp Arg His Pro His Phe Pro Thr Arg Tyr Tyr 20 25 30Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 35 40
45Thr Val Pro Leu Gln Pro Pro Thr Ala Thr Ile Ser Gly Leu Lys Pro
50 55 60Gly Val Asp Tyr Thr Ile Thr Gly Tyr Ala Val Thr Val Ala Gln
Asn65 70 75 80Asp His Glu Leu Ile Thr Pro Ile Ser Ile Asn Tyr Arg
Thr 85 902295PRTArtificial Sequencebinding polypeptide 22Val Ser
Asp Val Pro Arg Asp Leu Gln Glu Val Val Ala Ala Thr Pro 1 5 10
15Thr Ser Leu Leu Ile Ser Trp Arg His Pro His Phe Pro Thr Arg Tyr
20 25 30Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln
Glu 35 40 45Phe Thr Val Pro Leu Gln Pro Pro Ala Ala Thr Ile Ser Gly
Leu Lys 50 55 60Pro Gly Val Asp Tyr Thr Ile Thr Gly Tyr Ala Val Thr
Met Ala Gln65 70 75 80Ser Gly His Glu Leu Phe Thr Pro Ile Ser Ile
Asn Tyr Arg Thr 85 90 952386PRTArtificial Sequencebinding
polypeptide 23Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser
Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr
Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val
Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly
Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr Val Glu Arg Asn
Gly Arg Lys Leu Met Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
852486PRTArtificial Sequencebinding polypeptide 24Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Val Glu Arg Asn Gly Arg Val Leu Met Thr Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 852586PRTArtificial
Sequencebinding polypeptide 25Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Val Glu Arg Asn Gly Arg His Leu Met Thr Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 852686PRTArtificial Sequencebinding polypeptide
26Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1
5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro
Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr
Ile Thr Gly 50 55 60Tyr Ala Val Thr Val Glu Arg Asn Gly Arg Met Leu
Met Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
852786PRTArtificial Sequencebinding polypeptide 27Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Leu Glu Arg Asn Gly Arg Val Leu Met Thr Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 852886PRTArtificial
Sequencebinding polypeptide 28Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Leu Glu Arg Asn Gly Arg Val Leu Asn Thr Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 852986PRTArtificial Sequencebinding polypeptide
29Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1
5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro
Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr
Ile Thr Gly 50 55 60Tyr Ala Val Thr Val Glu Arg Asn Gly Arg Gln Leu
Met Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
853086PRTArtificial Sequencebinding polypeptide 30Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Val Glu Arg Asn Gly Arg Thr Leu Phe Thr Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg
Thr 853186PRTArtificial Sequencebinding polypeptide 31Glu Val Val
Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro
His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25
30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr
35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr
Gly 50 55 60Tyr Ala Val Thr Leu Glu Arg Asn Gly Arg Glu Leu Met Thr
Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 853286PRTArtificial
Sequencebinding polypeptide 32Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Leu Glu Arg Asn Gly Arg Leu Leu Asn Thr Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 853386PRTArtificial Sequencebinding polypeptide
33Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1
5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro
Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr
Ile Thr Gly 50 55 60Tyr Ala Val Thr His Glu Arg Asn Gly Arg Val Leu
Met Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
853486PRTArtificial Sequencebinding polypeptide 34Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Glu Glu Arg Asn Gly Arg Val Leu Phe Thr Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 853586PRTArtificial
Sequencebinding polypeptide 35Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Val Glu Arg Asn Gly Arg Gln Leu Tyr Thr Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 853686PRTArtificial Sequencebinding polypeptide
36Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1
5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro
Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr
Ile Thr Gly 50 55 60Tyr Ala Val Thr Val Glu Arg Asn Gly Arg Ala Leu
Met Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
853786PRTArtificial Sequencebinding polypeptide 37Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Val Glu Arg Asn Gly Arg Asn Leu Met Thr Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 853886PRTArtificial
Sequencebinding polypeptide 38Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Leu Glu Arg Asn Gly Arg Val Leu Ile Thr Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 853986PRTArtificial Sequencebinding polypeptide
39Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1
5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro
Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr
Ile Thr Gly 50 55 60Tyr Ala Val Thr Val Glu Arg Asn Gly Arg Val Leu
Asn Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
854086PRTArtificial Sequencebinding polypeptide 40Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Val Glu Arg Asn Gly Lys Val Leu Met Thr Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 854186PRTArtificial
Sequencebinding polypeptide 41Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Val Glu Arg Asn Gly Arg Thr Leu Met Met Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 854286PRTArtificial Sequencebinding polypeptide
42Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1
5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro
Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr
Ile Thr Gly 50 55 60Tyr Ala Val Thr Met Glu Arg Asn Gly Arg Glu Leu
Met Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
854386PRTArtificial Sequencebinding polypeptide 43Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Glu Glu Arg Asn Gly Arg Thr Leu Arg Thr Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 854486PRTArtificial
Sequencebinding polypeptide 44Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Val Glu Arg Asn Gly Lys Thr Leu Met Thr Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 854586PRTArtificial Sequencebinding polypeptide
45Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1
5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro
Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr
Ile Thr Gly 50 55 60Tyr Ala Val Thr Leu Glu Arg Asn Asp Arg Val Leu
Leu Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
854686PRTArtificial Sequencebinding polypeptide 46Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Leu Glu Arg Asn Gly Arg Lys Leu Met Thr Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 854786PRTArtificial
Sequencebinding polypeptide 47Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Val Glu Pro Asn Gly Arg Val Leu Asn Thr Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 854886PRTArtificial Sequencebinding polypeptide
48Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1
5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro
Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr
Ile Thr Gly 50 55 60Tyr Ala Val Thr Val Glu Arg Asn Asp Arg Val Leu
Phe Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
854986PRTArtificial Sequencebinding polypeptide 49Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Val Glu Arg Asn Gly Arg Glu Leu Lys Thr Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 855086PRTArtificial
Sequencebinding polypeptide 50Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Val Glu Arg Asn Gly Arg Glu Leu Arg Thr Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 855186PRTArtificial Sequencebinding polypeptide
51Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1
5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro
Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr
Ile Thr Gly 50 55 60Tyr Ala Val Thr Gln Glu Arg Asn Gly Arg Glu Leu
Met Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
855286PRTArtificial Sequencebinding polypeptide 52Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Val Glu Arg Asn Gly Arg Glu Leu Met Thr Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 855386PRTArtificial
Sequencebinding polypeptide 53Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Val Glu Arg Asn Gly Arg Val Leu Ser Val Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 855486PRTArtificial Sequencebinding polypeptide
54Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1
5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro
Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr
Ile Thr Gly 50 55 60Tyr Ala Val Thr Val Glu Arg Asp Gly Arg Thr Leu
Arg Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
855586PRTArtificial Sequencebinding polypeptide 55Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Val Glu Arg Asn Gly Arg Glu Leu Asn Thr Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 855686PRTArtificial
Sequencebinding polypeptide 56Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Val Glu Arg Asn Gly Arg Val Leu Ile Val Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 855786PRTArtificial Sequencebinding polypeptide
57Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1
5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro
Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr
Ile Thr Gly 50 55 60Tyr Ala Val Thr Leu Glu Arg Asn Gly Arg Glu Leu
Met Val Pro Ile65
70 75 80Ser Ile Asn Tyr Arg Thr 855886PRTArtificial Sequencebinding
polypeptide 58Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser
Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr
Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val
Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly
Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr Asp Gly Arg Asn
Asp Arg Lys Leu Met Val Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
855986PRTArtificial Sequencebinding polypeptide 59Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Val Glu His Asn Gly Arg Thr Ser Phe Thr Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 856086PRTArtificial
Sequencebinding polypeptide 60Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Val Glu Arg Asp Gly Arg Lys Leu Tyr Thr Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 856186PRTArtificial Sequencebinding polypeptide
61Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1
5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro
Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr
Ile Thr Gly 50 55 60Tyr Ala Val Thr Leu Glu Arg Asn Gly Arg Glu Leu
Asn Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
856286PRTArtificial Sequencebinding polypeptide 62Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Asp Gly Trp Asn Gly Arg Leu Leu Ser Ile Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 856386PRTArtificial
Sequencebinding polypeptide 63Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Asp Gly Gln Asn Gly Arg Leu Leu Asn Val Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 856486PRTArtificial Sequencebinding polypeptide
64Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1
5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro
Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr
Ile Thr Gly 50 55 60Tyr Ala Val Thr Ile Glu Lys Asn Gly Arg His Leu
Asn Ile Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
856586PRTArtificial Sequencebinding polypeptide 65Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Asp Gly Trp Asn Gly Lys Met Leu Ser Val Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 856686PRTArtificial
Sequencebinding polypeptide 66Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Asp Gly Tyr Asn Asp Arg Leu Leu Phe Ile Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 856786PRTArtificial Sequencebinding polypeptide
67Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1
5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro
Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr
Ile Thr Gly 50 55 60Tyr Ala Val Thr Asp Gly Pro Asn Asp Arg Leu Leu
Asn Ile Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
856886PRTArtificial Sequencebinding polypeptide 68Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Asp Gly Pro Asn Asn Arg Glu Leu Ile Val Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 856986PRTArtificial
Sequencebinding polypeptide 69Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Asp Gly Leu Asn Gly Lys Tyr Leu Phe Val Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 857086PRTArtificial Sequencebinding polypeptide
70Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1
5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro
Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr
Ile Thr Gly 50 55 60Tyr Ala Val Thr Glu Gly Trp Asn Asp Arg Glu Leu
Phe Val Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
857186PRTArtificial Sequencebinding polypeptide 71Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Phe Gly Trp Asn Gly Arg Glu Leu Leu Thr Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 857286PRTArtificial
Sequencebinding polypeptide 72Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Phe Gly Trp Asn Asp Arg Glu Leu Leu Ile Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 857386PRTArtificial Sequencebinding polypeptide
73Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1
5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro
Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr
Ile Thr Gly 50 55 60Tyr Ala Val Thr Leu Glu Trp Asn Asn Arg Val Leu
Met Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
857486PRTArtificial Sequencebinding polypeptide 74Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Val Glu Trp Asn Gly Arg Val Leu Met Thr Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 857586PRTArtificial
Sequencebinding polypeptide 75Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Asn Glu Arg Asn Gly Arg Glu Leu Met Thr Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 857686PRTArtificial Sequencebinding polypeptide
76Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1
5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro
Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr
Ile Thr Gly 50 55 60Tyr Ala Val Thr Leu Glu Arg Asn Gly Lys Glu Leu
Met Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
857786PRTArtificial Sequencebinding polypeptide 77Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Val Glu Arg Asn Gly Arg Glu Leu Leu Thr Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 857886PRTArtificial
Sequencebinding polypeptide 78Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Val Glu Arg Asn Gly Arg Glu Leu Lys Thr Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 857986PRTArtificial Sequencebinding polypeptide
79Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1
5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro
Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr
Ile Thr Gly 50 55 60Tyr Ala Val Thr Gln Glu Arg Asn Gly Arg Glu Leu
Arg Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
858086PRTArtificial Sequencebinding polypeptide 80Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Val Glu Arg Asn Gly Arg Glu Leu Leu Trp Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 858186PRTArtificial
Sequencebinding polypeptide 81Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Leu Glu Arg Asn Gly Arg Glu Leu Met Ile Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 858286PRTArtificial Sequencebinding polypeptide
82Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1
5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro
Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr
Ile Thr Gly 50 55 60Tyr Ala Val Thr Val Glu Arg Asn Gly Leu Val Leu
Met Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
858386PRTArtificial Sequencebinding polypeptide 83Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Val Glu Arg Asn Gly Arg Val Leu Ile Ile Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 858486PRTArtificial
Sequencebinding polypeptide 84Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55
60Tyr Ala Val Thr Val Glu Arg Asn Gly His Lys Leu Phe Thr Pro Ile65
70 75 80Ser Ile Asn Tyr Arg Thr 858586PRTArtificial Sequencebinding
polypeptide 85Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser
Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr
Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val
Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly
Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr Val Glu Arg Asn
Glu Arg Val Leu Met Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
858686PRTArtificial Sequencebinding polypeptide 86Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Phe Gly Pro Asn Asp Arg Glu Leu Leu Thr Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 858786PRTArtificial
Sequencebinding polypeptide 87Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Met Gly Pro Asn Asp Arg Glu Leu Leu Thr Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 858886PRTArtificial Sequencebinding polypeptide
88Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1
5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro
Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr
Ile Thr Gly 50 55 60Tyr Ala Val Thr Met Gly Lys Asn Asp Arg Glu Leu
Leu Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
858987PRTArtificial Sequencebinding polypeptide 89Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15His Pro
His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr 20 25 30Gly
Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro 35 40
45Thr Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr
50 55 60Gly Tyr Ala Val Thr Val His Trp Asn Gly Arg Glu Leu Met Thr
Pro65 70 75 80Ile Ser Ile Asn Tyr Arg Thr 859086PRTArtificial
Sequencebinding polypeptide 90Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Glu Glu Trp Asn Gly Arg Val Leu Met Thr Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 859186PRTArtificial Sequencebinding polypeptide
91Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1
5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro
Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr
Ile Thr Gly 50 55 60Tyr Ala Val Thr Val Glu Arg Asn Gly His Thr Leu
Met Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
859286PRTArtificial Sequencebinding polypeptide 92Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Val Glu Glu Asn Gly Arg Gln Leu Met Thr Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 859386PRTArtificial
Sequencebinding polypeptide 93Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Leu Glu Arg Asn Gly Gln Val Leu Phe Thr Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 859486PRTArtificial Sequencebinding polypeptide
94Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1
5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro
Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr
Ile Thr Gly 50 55 60Tyr Ala Val Thr Val Glu Arg Asn Gly Gln Val Leu
Tyr Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
859586PRTArtificial Sequencebinding polypeptide 95Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Trp Gly Tyr Lys Asp His Glu Leu Leu Ile Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 859686PRTArtificial
Sequencebinding polypeptide 96Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Leu Gly Arg Asn Asp Arg Glu Leu Leu Thr Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 859786PRTArtificial Sequencebinding polypeptide
97Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1
5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro
Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr
Ile Thr Gly 50 55 60Tyr Ala Val Thr Asp Gly Pro Asn Asp Arg Leu Leu
Asn Ile Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
859886PRTArtificial Sequencebinding polypeptide 98Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Phe Ala Arg Asp Gly His Glu Ile Leu Thr Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 859986PRTArtificial
Sequencebinding polypeptide 99Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Leu Glu Gln Asn Gly Arg Glu Leu Met Thr Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 8510086PRTArtificial Sequencebinding polypeptide
100Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His
1 5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln
Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr
Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr Val Glu Glu Asn Gly Arg Val
Leu Asn Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
8510186PRTArtificial Sequencebinding polypeptide 101Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Leu Glu Pro Asn Gly Arg Tyr Leu Met Val Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 8510286PRTArtificial
Sequencebinding polypeptide 102Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Glu Gly Arg Asn Gly Arg Glu Leu Phe Ile Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 8510386PRTArtificial Sequencebinding polypeptide
103Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His
1 5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln
Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr
Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr Ser Gly Arg Asn Asp Arg Glu
Leu Leu Val Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
8510486PRTArtificial Sequencebinding polypeptide 104Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Val Glu Arg Asp Gly Arg Glu Leu Asn Ile Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 8510586PRTArtificial
Sequencebinding polypeptide 105Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Val Glu Gln Asn Gly Arg Val Leu Met Thr Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 8510686PRTArtificial Sequencebinding polypeptide
106Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His
1 5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln
Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr
Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr Val Glu His Asn Gly Arg Val
Leu Asn Ile Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
8510786PRTArtificial Sequencebinding polypeptide 107Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Met Ala Pro Asn Gly Arg Glu Leu Leu Thr Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 8510886PRTArtificial
Sequencebinding polypeptide 108Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Val Glu Gln Asn Gly Arg Val Leu Asn Thr Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 8510986PRTArtificial Sequencebinding polypeptide
109Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His
1 5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln
Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr
Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr Asp Gly Arg Asn Gly His Glu
Leu Met Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
8511086PRTArtificial Sequencebinding polypeptide 110Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Glu Gly Arg Asn Gly Arg Glu Leu Met Val Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 8511186PRTArtificial
Sequencebinding polypeptide 111Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro
Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr Leu Glu Arg
Asn Asn Arg Glu Leu Leu Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg
Thr 8511286PRTArtificial Sequencebinding polypeptide 112Glu Val Val
Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro
His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25
30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr
35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr
Gly 50 55 60Tyr Ala Val Thr Met Glu Arg Ser Gly Arg Glu Leu Met Thr
Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 8511394PRTArtificial
Sequencebinding polypeptide 113Val Ser Asp Val Pro Arg Asp Leu Glu
Val Val Ala Ala Thr Pro Thr 1 5 10 15Ser Leu Leu Ile Ser Trp Arg
His Pro His Phe Pro Thr Arg Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 35 40 45Thr Val Pro Leu Gln
Pro Pro Ala Ala Thr Ile Ser Gly Leu Lys Pro 50 55 60Gly Val Asp Tyr
Thr Ile Thr Gly Tyr Ala Val Thr Arg Ala Leu Leu65 70 75 80Ser Ile
Glu Leu Phe Thr Pro Ile Ser Ile Asn Tyr Arg Thr 85
9011494PRTArtificial Sequencebinding polypeptide 114Val Ser Asp Val
Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr 1 5 10 15Ser Leu
Leu Ile Ser Trp Arg His Pro His Phe Pro Thr Arg Tyr Tyr 20 25 30Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 35 40
45Thr Val Pro Leu Gln Pro Pro Ala Ala Thr Ile Ser Gly Leu Lys Pro
50 55 60Gly Val Asp Tyr Thr Ile Thr Gly Tyr Ala Val Thr Phe Ala Arg
Lys65 70 75 80Gly Thr Glu Leu Phe Thr Pro Ile Ser Ile Asn Tyr Arg
Thr 85 9011594PRTArtificial Sequencebinding polypeptide 115Val Ser
Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr 1 5 10
15Ser Leu Leu Ile Ser Trp Arg His Pro His Phe Pro Thr Arg Tyr Tyr
20 25 30Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu
Phe 35 40 45Thr Val Pro Leu Gln Pro Pro Ala Ala Thr Ile Ser Gly Leu
Lys Pro 50 55 60Gly Val Asp Tyr Thr Ile Thr Gly Tyr Ala Val Thr Leu
Glu Arg Cys65 70 75 80Gly Arg Glu Leu Phe Thr Pro Ile Ser Ile Asn
Tyr Arg Thr 85 9011694PRTArtificial Sequencebinding polypeptide
116Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr
1 5 10 15Ser Leu Leu Ile Ser Trp Arg His Pro His Phe Pro Thr Arg
Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val
Gln Glu Phe 35 40 45Thr Val Pro Leu Gln Pro Pro Ala Ala Thr Ile Ser
Gly Leu Lys Pro 50 55 60Gly Val Asp Tyr Thr Ile Thr Gly Tyr Ala Val
Thr Arg Glu Arg Asn65 70 75 80Gly Arg Glu Leu Phe Thr Pro Ile Ser
Ile Asn Tyr Arg Thr 85 9011794PRTArtificial Sequencebinding
polypeptide 117Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala
Thr Pro Thr 1 5 10 15Ser Leu Leu Ile Ser Trp Arg His Pro His Phe
Pro Thr Arg Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn
Ser Pro Val Gln Glu Phe 35 40 45Thr Val Pro Leu Gln Pro Pro Ala Ala
Thr Ile Ser Gly Leu Lys Pro 50 55 60Gly Val Asp Tyr Thr Ile Thr Gly
Tyr Ala Val Thr Lys Glu Arg Asn65 70 75 80Gly Arg Glu Leu Phe Thr
Pro Ile Ser Ile Asn Tyr Arg Thr 85 9011894PRTArtificial
Sequencebinding polypeptide 118Val Ser Asp Val Pro Arg Asp Leu Glu
Val Val Ala Ala Thr Pro Thr 1 5 10 15Ser Leu Leu Ile Ser Trp Arg
His Pro His Phe Pro Thr Arg Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 35 40 45Thr Val Pro Leu Gln
Pro Pro Ala Ala Thr Ile Ser Gly Leu Lys Pro 50 55 60Gly Val Asp Tyr
Thr Ile Thr Gly Tyr Ala Val Thr Cys Glu Arg Asn65 70 75 80Gly Arg
Glu Leu Phe Thr Pro Ile Ser Ile Asn Tyr Arg Thr 85
9011994PRTArtificial Sequencebinding polypeptide 119Val Ser Asp Val
Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr 1 5 10 15Ser Leu
Leu Ile Ser Trp Arg His Pro His Phe Pro Thr Arg Tyr Tyr 20 25 30Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 35 40
45Thr Val Pro Leu Gln Pro Pro Ala Ala Thr Ile Ser Gly Leu Lys Pro
50 55 60Gly Val Asp Tyr Thr Ile Thr Gly Tyr Ala Val Thr Leu Glu Arg
Thr65 70 75 80Gly Arg Glu Leu Phe Thr Pro Ile Ser Ile Asn Tyr Arg
Thr 85 9012094PRTArtificial Sequencebinding polypeptide 120Val Ser
Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr 1 5 10
15Ser Leu Leu Ile Ser Trp Arg His Pro His Phe Pro Thr Arg Tyr Tyr
20 25 30Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu
Phe 35 40 45Thr Val Pro Leu Gln Pro Pro Ala Ala Thr Ile Ser Gly Leu
Lys Pro 50 55 60Gly Val Asp Tyr Thr Ile Thr Gly Tyr Ala Val Thr Trp
Glu Arg Thr65 70 75 80Gly Lys Glu Leu Phe Thr Pro Ile Ser Ile Asn
Tyr Arg Thr 85 9012194PRTArtificial Sequencebinding polypeptide
121Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr
1 5 10 15Ser Leu Leu Ile Ser Trp Arg His Pro His Phe Pro Thr Arg
Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val
Gln Glu Phe 35 40 45Thr Val Pro Leu Gln Pro Pro Ala Ala Thr Ile Ser
Gly Leu Lys Pro 50 55 60Gly Val Asp Tyr Thr Ile Thr Gly Tyr Ala Val
Thr Ile Glu Arg Thr65 70 75 80Cys Arg Glu Leu Phe Thr Pro Ile Ser
Ile Asn Tyr Arg Thr 85 9012294PRTArtificial Sequencebinding
polypeptide 122Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala
Thr Pro Thr 1 5 10 15Ser Leu Leu Ile Ser Trp Arg His Pro His Phe
Pro Thr Arg Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn
Ser Pro Val Gln Glu Phe 35 40 45Thr Val Pro Leu Gln Pro Pro Ala Ala
Thr Ile Ser Gly Leu Lys Pro 50 55 60Gly Val Asp Tyr Thr Ile Thr Gly
Tyr Ala Val Thr Gly Gly Met Ile65 70 75 80Val Arg Glu Leu Phe Thr
Pro Ile Ser Ile Asn Tyr Arg Thr 85 9012394PRTArtificial
Sequencebinding polypeptide 123Val Ser Asp Val Pro Arg Asp Leu Glu
Val Val Ala Ala Thr Pro Thr 1 5 10 15Ser Leu Leu Ile Ser Trp Arg
His Pro His Phe Pro Thr Arg Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 35 40 45Thr Val Pro Leu Gln
Pro Pro Ala Ala Thr Ile Ser Gly Leu Lys Pro 50 55 60Gly Val Asp Tyr
Thr Ile Thr Gly Tyr Ala Val Thr Phe Gly Arg Ser65 70 75 80Ser Arg
Glu Leu Phe Thr Pro Ile Ser Ile Asn Tyr Arg Thr 85
9012494PRTArtificial Sequencebinding polypeptide 124Val Ser Asp Val
Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr 1 5 10 15Ser Leu
Leu Ile Ser Trp Arg His Pro His Phe Pro Thr Arg Tyr Tyr 20 25 30Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 35 40
45Thr Val Pro Leu Gln Pro Pro Ala Ala Thr Ile Ser Gly Leu Lys Pro
50 55 60Gly Val Asp Tyr Thr Ile Thr Gly Tyr Ala Val Thr Arg His Lys
Ser65 70 75 80Arg Gly Glu Leu Phe Thr Pro Ile Ser Ile Asn Tyr Arg
Thr 85 9012594PRTArtificial Sequencebinding polypeptide 125Val Ser
Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr 1 5 10
15Ser Leu Leu Ile Ser Trp Arg His Pro His Phe Pro Thr Arg Tyr Tyr
20 25 30Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu
Phe 35 40 45Thr Val Pro Leu Gln Pro Pro Ala Ala Thr Ile Ser Gly Leu
Lys Pro 50 55 60Gly Val Asp Tyr Thr Ile Thr Gly Tyr Ala Val Thr Arg
His Arg Asp65 70 75 80Lys Arg Glu Leu Phe Thr Pro Ile Ser Ile Asn
Tyr Arg Thr 85 9012694PRTArtificial Sequencebinding polypeptide
126Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr
1 5 10 15Ser Leu Leu Ile Ser Trp Arg His Pro His Phe Pro Thr Arg
Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val
Gln Glu Phe 35 40 45Thr Val Pro Leu Gln Pro Pro Ala Ala Thr Ile Ser
Gly Leu Lys Pro 50 55 60Gly Val Asp Tyr Thr Ile Thr Gly Tyr Ala Val
Thr Tyr His Arg Gly65 70 75 80Arg Gly Glu Leu Phe Thr Pro Ile Ser
Ile Asn Tyr Arg Thr 85 9012794PRTArtificial Sequencebinding
polypeptide 127Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala
Thr Pro Thr 1 5 10 15Ser Leu Leu Ile Ser Trp Arg His Pro His Phe
Pro Thr Arg Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn
Ser Pro Val Gln Glu Phe 35 40 45Thr Val Pro Leu Gln Pro Pro Ala Ala
Thr Ile Ser Gly Leu Lys Pro 50 55 60Gly Val Asp Tyr Thr Ile Thr Gly
Tyr Ala Val Thr Arg His Arg Gly65 70 75 80Cys Arg Glu Leu Phe Thr
Pro Ile Ser Ile Asn Tyr Arg Thr 85 9012894PRTArtificial
Sequencebinding polypeptide 128Val Ser Asp Val Pro Arg Asp Leu Glu
Val Val Ala Ala Thr Pro Thr 1 5 10 15Ser Leu Leu Ile Ser Trp Arg
His Pro His Phe Pro Thr Arg Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 35 40 45Thr Val Pro Leu Gln
Pro Pro Ala Ala Thr Ile Ser Gly Leu Lys Pro 50 55 60Gly Val Asp Tyr
Thr Ile Thr Gly Tyr Ala Val Thr Ser His Arg Leu65 70 75 80Arg Lys
Glu Leu Phe Thr Pro Ile Ser Ile Asn Tyr Arg Thr 85
9012994PRTArtificial Sequencebinding polypeptide 129Val Ser Asp Val
Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr 1 5 10 15Ser Leu
Leu Ile Ser Trp Arg His Pro His Phe Pro Thr Arg Tyr Tyr 20 25 30Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 35 40
45Thr Val Pro Leu Gln Pro Pro Ala Ala Thr Ile Ser Gly Leu Lys Pro
50 55 60Gly Val Asp Tyr Thr Ile Thr Gly Tyr Ala Val Thr Met His Arg
Gln65 70 75 80Arg Gly Glu Leu Phe Thr Pro Ile Ser Ile Asn Tyr Arg
Thr 85 9013094PRTArtificial Sequencebinding polypeptide 130Val Ser
Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr 1 5 10
15Ser Leu Leu Ile Ser Trp Arg His Pro His Phe Pro Thr Arg Tyr Tyr
20 25 30Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu
Phe 35 40 45Thr Val Pro Leu Gln Pro Pro Ala Ala Thr Ile Ser Gly Leu
Lys Pro 50 55 60Gly Val Asp Tyr Thr Ile Thr Gly Tyr Ala Val Thr Phe
His Arg Arg65 70 75 80Arg Gly Glu Leu Phe Thr Pro Ile Ser Ile Asn
Tyr Arg Thr 85 9013194PRTArtificial Sequencebinding polypeptide
131Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr
1 5 10 15Ser Leu Leu Ile Ser Trp Arg His Pro His Phe Pro Thr Arg
Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val
Gln Glu Phe 35 40 45Thr Val Pro Leu Gln Pro Pro Ala Ala Thr Ile Ser
Gly Leu Lys Pro 50 55 60Gly Val Asp Tyr Thr Ile Thr Gly Tyr Ala Val
Thr Phe His Arg Arg65 70 75 80Arg Gly Glu Leu Phe Thr Pro Ile Ser
Ile Asn Tyr Arg Thr 85 9013294PRTArtificial Sequencebinding
polypeptide 132Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala
Thr Pro Thr 1 5 10 15Ser Leu Leu Ile Ser Trp Arg His Pro His Phe
Pro Thr Arg Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn
Ser Pro Val Gln Glu Phe 35 40 45Thr Val Pro Leu Gln Pro Pro Ala Ala
Thr Ile Ser Gly Leu Lys Pro 50 55 60Gly Val Asp Tyr Thr Ile Thr Gly
Tyr Ala Val Thr Ser His Arg Arg65 70 75 80Arg Asn Glu Leu Phe Thr
Pro Ile Ser Ile Asn Tyr Arg Thr 85 9013394PRTArtificial
Sequencebinding polypeptide 133Val Ser Asp Val Pro Arg Asp Leu Glu
Val Val Ala Ala Thr Pro Thr 1 5 10 15Ser Leu Leu Ile Ser Trp Arg
His Pro His Phe Pro Thr Arg Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 35 40 45Thr Val Pro Leu Gln
Pro Pro Ala Ala Thr Ile Ser Gly Leu Lys Pro 50 55 60Gly Val Asp Tyr
Thr Ile Thr Gly Tyr Ala Val Thr Leu His Arg Arg65 70 75 80Val Arg
Glu Leu Phe Thr Pro Ile Ser Ile Asn Tyr Arg Thr 85
9013494PRTArtificial Sequencebinding polypeptide 134Val Ser Asp Val
Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr 1 5 10 15Ser Leu
Leu Ile Ser Trp Arg His Pro His Phe Pro Thr Arg Tyr Tyr 20 25 30Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 35 40
45Thr Val Pro Leu Gln Pro Pro Ala Ala Thr Ile Ser Gly Leu Lys Pro
50 55 60Gly Val Asp Tyr Thr Ile Thr Gly Tyr Ala Val Thr Arg His Arg
Arg65 70 75 80Arg Gly Glu Leu Phe Thr Pro Ile Ser Ile Asn Tyr Arg
Thr 85 9013594PRTArtificial Sequencebinding polypeptide 135Val Ser
Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr 1 5 10
15Ser Leu Leu Ile Ser Trp Arg His Pro His Phe Pro Thr Arg Tyr Tyr
20 25 30Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu
Phe 35 40 45Thr Val Pro Leu Gln Pro Pro Ala Ala Thr Ile Ser Gly Leu
Lys Pro 50 55 60Gly Val Asp Tyr Thr Ile Thr Gly Tyr Ala Val Thr Trp
His Arg Ser65 70 75 80Arg Lys Glu Leu Phe Thr Pro Ile Ser Ile Asn
Tyr Arg Thr 85 9013694PRTArtificial Sequencebinding polypeptide
136Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr
1 5 10 15Ser Leu Leu Ile Ser Trp Arg His Pro His Phe Pro Thr Arg
Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val
Gln Glu Phe 35 40 45Thr Val Pro Leu Gln Pro Pro Ala Ala Thr Ile Ser
Gly Leu Lys Pro 50 55 60Gly Val Asp Tyr Thr Ile Thr Gly Tyr Ala Val
Thr Arg His Arg Ser65 70
75 80Arg Gly Glu Leu Phe Thr Pro Ile Ser Ile Asn Tyr Arg Thr 85
9013794PRTArtificial Sequencebinding polypeptide 137Val Ser Asp Val
Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr 1 5 10 15Ser Leu
Leu Ile Ser Trp Arg His Pro His Phe Pro Thr Arg Tyr Tyr 20 25 30Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 35 40
45Thr Val Pro Leu Gln Pro Pro Ala Ala Thr Ile Ser Gly Leu Lys Pro
50 55 60Gly Val Asp Tyr Thr Ile Thr Gly Tyr Ala Val Thr Val His Arg
Thr65 70 75 80Gly Arg Glu Leu Phe Thr Pro Ile Ser Ile Asn Tyr Arg
Thr 85 9013894PRTArtificial Sequencebinding polypeptide 138Val Ser
Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr 1 5 10
15Ser Leu Leu Ile Ser Trp Arg His Pro His Phe Pro Thr Arg Tyr Tyr
20 25 30Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu
Phe 35 40 45Thr Val Pro Leu Gln Pro Pro Ala Ala Thr Ile Ser Gly Leu
Lys Pro 50 55 60Gly Val Asp Tyr Thr Ile Thr Gly Tyr Ala Val Thr Trp
His Arg Val65 70 75 80Arg Gly Glu Leu Phe Thr Pro Ile Ser Ile Asn
Tyr Arg Thr 85 9013994PRTArtificial Sequencebinding polypeptide
139Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr
1 5 10 15Ser Leu Leu Ile Ser Trp Arg His Pro His Phe Pro Thr Arg
Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val
Gln Glu Phe 35 40 45Thr Val Pro Leu Gln Pro Pro Ala Ala Thr Ile Ser
Gly Leu Lys Pro 50 55 60Gly Val Asp Tyr Thr Ile Thr Gly Tyr Ala Val
Thr Trp His Arg Val65 70 75 80Arg Gly Glu Leu Phe Thr Pro Ile Ser
Ile Asn Tyr Arg Thr 85 9014094PRTArtificial Sequencebinding
polypeptide 140Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala
Thr Pro Thr 1 5 10 15Ser Leu Leu Ile Ser Trp Arg His Pro His Phe
Pro Thr Arg Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn
Ser Pro Val Gln Glu Phe 35 40 45Thr Val Pro Leu Gln Pro Pro Ala Ala
Thr Ile Ser Gly Leu Lys Pro 50 55 60Gly Val Asp Tyr Thr Ile Thr Gly
Tyr Ala Val Thr Trp His Arg Trp65 70 75 80Arg Gly Glu Leu Phe Thr
Pro Ile Ser Ile Asn Tyr Arg Thr 85 9014194PRTArtificial
Sequencebinding polypeptide 141Val Ser Asp Val Pro Arg Asp Leu Glu
Val Val Ala Ala Thr Pro Thr 1 5 10 15Ser Leu Leu Ile Ser Trp Arg
His Pro His Phe Pro Thr Arg Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 35 40 45Thr Val Pro Leu Gln
Pro Pro Ala Ala Thr Ile Ser Gly Leu Lys Pro 50 55 60Gly Val Asp Tyr
Thr Ile Thr Gly Tyr Ala Val Thr Trp Lys Arg Ser65 70 75 80Gly Gly
Glu Leu Phe Thr Pro Ile Ser Ile Asn Tyr Arg Thr 85
9014294PRTArtificial Sequencebinding polypeptide 142Val Ser Asp Val
Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr 1 5 10 15Ser Leu
Leu Ile Ser Trp Arg His Pro His Phe Pro Thr Arg Tyr Tyr 20 25 30Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 35 40
45Thr Val Pro Leu Gln Pro Pro Ala Ala Thr Ile Ser Gly Leu Lys Pro
50 55 60Gly Val Asp Tyr Thr Ile Thr Gly Tyr Ala Val Thr Arg Leu Xaa
Asn65 70 75 80Xaa Val Glu Leu Phe Thr Pro Ile Ser Ile Asn Tyr Arg
Thr 85 9014394PRTArtificial Sequencebinding polypeptide 143Val Ser
Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr 1 5 10
15Ser Leu Leu Ile Ser Trp Arg His Pro His Phe Pro Thr Arg Tyr Tyr
20 25 30Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu
Phe 35 40 45Thr Val Pro Leu Gln Pro Pro Ala Ala Thr Ile Ser Gly Leu
Lys Pro 50 55 60Gly Val Asp Tyr Thr Ile Thr Gly Tyr Ala Val Thr Trp
Arg Thr Pro65 70 75 80His Ala Glu Leu Phe Thr Pro Ile Ser Ile Asn
Tyr Arg Thr 85 9014494PRTArtificial Sequencebinding polypeptide
144Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr
1 5 10 15Ser Leu Leu Ile Ser Trp Arg His Pro His Phe Pro Thr Arg
Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val
Gln Glu Phe 35 40 45Thr Val Pro Leu Gln Pro Pro Ala Ala Thr Ile Ser
Gly Leu Lys Pro 50 55 60Gly Val Asp Tyr Thr Ile Thr Gly Tyr Ala Val
Thr Leu Ser Pro His65 70 75 80Ser Val Glu Leu Phe Thr Pro Ile Ser
Ile Asn Tyr Arg Thr 85 9014594PRTArtificial Sequencebinding
polypeptide 145Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala
Thr Pro Thr 1 5 10 15Ser Leu Leu Ile Ser Trp Arg His Pro His Phe
Pro Thr Arg Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn
Ser Pro Val Gln Glu Phe 35 40 45Thr Val Pro Leu Gln Pro Pro Ala Ala
Thr Ile Ser Gly Leu Lys Pro 50 55 60Gly Val Asp Tyr Thr Ile Thr Gly
Tyr Ala Val Thr Val Ser Arg Gln65 70 75 80Lys Ala Glu Leu Phe Thr
Pro Ile Ser Ile Asn Tyr Arg Thr 85 9014694PRTArtificial
Sequencebinding polypeptide 146Val Ser Asp Val Pro Arg Asp Leu Glu
Val Val Ala Ala Thr Pro Thr 1 5 10 15Ser Leu Leu Ile Ser Trp Arg
His Pro His Phe Pro Thr Arg Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 35 40 45Thr Val Pro Leu Gln
Pro Pro Ala Ala Thr Ile Ser Gly Leu Lys Pro 50 55 60Gly Val Asp Tyr
Thr Ile Thr Gly Tyr Ala Val Thr Ser Ser Tyr Ser65 70 75 80Lys Leu
Glu Leu Phe Thr Pro Ile Ser Ile Asn Tyr Arg Thr 85
9014794PRTArtificial Sequencebinding polypeptide 147Val Ser Asp Val
Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr 1 5 10 15Ser Leu
Leu Ile Ser Trp Arg His Pro His Phe Pro Thr Arg Tyr Tyr 20 25 30Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 35 40
45Thr Val Pro Leu Gln Pro Pro Ala Ala Thr Ile Ser Gly Leu Lys Pro
50 55 60Gly Val Asp Tyr Thr Ile Thr Gly Tyr Ala Val Thr Leu Thr Asp
Arg65 70 75 80Gly Ser Glu Leu Phe Thr Pro Ile Ser Ile Asn Tyr Arg
Thr 85 9014894PRTArtificial Sequencebinding polypeptide 148Val Ser
Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr 1 5 10
15Ser Leu Leu Ile Ser Trp Arg His Pro His Phe Pro Thr Arg Tyr Tyr
20 25 30Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu
Phe 35 40 45Thr Val Pro Leu Gln Pro Pro Ala Ala Thr Ile Ser Gly Leu
Lys Pro 50 55 60Gly Val Asp Tyr Thr Ile Thr Gly Tyr Ala Val Thr Gly
Thr Arg Thr65 70 75 80Arg Ser Glu Leu Phe Thr Pro Ile Ser Ile Asn
Tyr Arg Thr 85 9014994PRTArtificial Sequencebinding polypeptide
149Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr
1 5 10 15Ser Leu Leu Ile Ser Trp Arg His Pro His Phe Pro Thr Arg
Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val
Gln Glu Phe 35 40 45Thr Val Pro Leu Gln Pro Pro Ala Ala Thr Ile Ser
Gly Leu Lys Pro 50 55 60Gly Val Asp Tyr Thr Ile Thr Gly Tyr Ala Val
Thr Pro Val Ala Gly65 70 75 80Cys Ser Glu Leu Phe Thr Pro Ile Ser
Ile Asn Tyr Arg Thr 85 9015094PRTArtificial Sequencebinding
polypeptide 150Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala
Thr Pro Thr 1 5 10 15Ser Leu Leu Ile Ser Trp Arg His Pro His Phe
Pro Thr Arg Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn
Ser Pro Val Gln Glu Phe 35 40 45Thr Val Pro Leu Gln Pro Pro Ala Ala
Thr Ile Ser Gly Leu Lys Pro 50 55 60Gly Val Asp Tyr Thr Ile Thr Gly
Tyr Ala Val Thr Trp Trp Gln Thr65 70 75 80Pro Arg Glu Leu Phe Thr
Pro Ile Ser Ile Asn Tyr Arg Thr 85 9015194PRTArtificial
Sequencebinding polypeptide 151Val Ser Asp Val Pro Arg Asp Leu Glu
Val Val Ala Ala Thr Pro Thr 1 5 10 15Ser Leu Leu Ile Ser Trp Arg
His Pro His Phe Pro Thr Arg Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 35 40 45Thr Val Pro Leu Gln
Pro Pro Ala Ala Thr Ile Ser Gly Leu Lys Pro 50 55 60Gly Val Asp Tyr
Thr Ile Thr Gly Tyr Ala Val Thr Trp Trp Gln Thr65 70 75 80Pro Arg
Glu Leu Phe Thr Pro Ile Ser Ile Asn Tyr Arg Thr 85
9015294PRTArtificial Sequencebinding polypeptide 152Val Ser Asp Val
Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr 1 5 10 15Ser Leu
Leu Ile Ser Trp Arg His Pro His Phe Pro Thr Arg Tyr Tyr 20 25 30Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 35 40
45Thr Val Pro Leu Gln Pro Pro Ala Ala Thr Ile Ser Gly Leu Lys Pro
50 55 60Gly Val Asp Tyr Thr Ile Thr Gly Tyr Ala Val Thr Trp Glu Arg
Asn65 70 75 80Gly Arg Glu Leu Phe Thr Pro Ile Ser Ile Asn Tyr Arg
Thr 85 9015394PRTArtificial Sequencebinding polypeptide 153Val Ser
Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr 1 5 10
15Ser Leu Leu Ile Ser Trp Arg His Pro His Phe Pro Thr Arg Tyr Tyr
20 25 30Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu
Phe 35 40 45Thr Val Pro Leu Gln Pro Pro Ala Ala Thr Ile Ser Gly Leu
Lys Pro 50 55 60Gly Val Asp Tyr Thr Ile Thr Gly Tyr Ala Val Thr Trp
Glu Trp Asn65 70 75 80Gly Arg Glu Leu Phe Thr Pro Ile Ser Ile Asn
Tyr Arg Thr 85 9015494PRTArtificial Sequencebinding polypeptide
154Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr
1 5 10 15Ser Leu Leu Ile Ser Trp Arg His Pro His Phe Pro Thr Arg
Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val
Gln Glu Phe 35 40 45Thr Val Pro Leu Gln Pro Pro Ala Ala Thr Ile Ser
Gly Leu Lys Pro 50 55 60Gly Val Asp Tyr Thr Ile Thr Gly Tyr Ala Val
Thr Lys Glu Arg Asn65 70 75 80Gly Arg Glu Leu Phe Thr Pro Ile Ser
Ile Asn Tyr Arg Thr 85 9015594PRTArtificial Sequencebinding
polypeptide 155Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala
Thr Pro Thr 1 5 10 15Ser Leu Leu Ile Ser Trp Leu His Pro His Phe
Pro Thr His Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn
Ser Pro Val Gln Glu Phe 35 40 45Thr Val Pro Leu Gln Pro Pro Ala Ala
Thr Ile Ser Gly Leu Lys Pro 50 55 60Gly Val Asp Tyr Thr Ile Thr Gly
Tyr Ala Val Thr Gly Ala Leu Asn65 70 75 80Thr Ser Glu Leu Phe Thr
Pro Ile Ser Ile Asn Tyr Arg Thr 85 9015694PRTArtificial
Sequencebinding polypeptide 156Val Ser Asp Val Pro Arg Asp Leu Glu
Val Val Ala Ala Thr Pro Thr 1 5 10 15Ser Leu Leu Ile Ser Trp Arg
His Pro His Phe Pro Thr His Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 35 40 45Thr Val Pro Leu Gln
Pro Pro Ala Ala Thr Ile Ser Gly Leu Lys Pro 50 55 60Gly Val Asp Tyr
Thr Ile Thr Gly Tyr Ala Val Thr Phe Gly Arg Glu65 70 75 80Arg Arg
Glu Leu Phe Thr Pro Ile Ser Ile Asn Tyr Arg Thr 85
9015794PRTArtificial Sequencebinding polypeptide 157Val Ser Asp Val
Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr 1 5 10 15Ser Leu
Leu Ile Ser Trp Leu His Pro His Phe Pro Thr His Tyr Tyr 20 25 30Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 35 40
45Thr Val Pro Leu Gln Pro Pro Ala Ala Thr Ile Ser Gly Leu Lys Pro
50 55 60Gly Val Asp Tyr Thr Ile Thr Gly Tyr Ala Val Thr Ser Gly Arg
Val65 70 75 80Ser Phe Glu Leu Phe Thr Pro Ile Ser Ile Asn Tyr Arg
Thr 85 9015894PRTArtificial Sequencebinding polypeptide 158Val Ser
Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr 1 5 10
15Ser Leu Leu Ile Ser Trp Arg His Pro His Phe Pro Thr His Tyr Tyr
20 25 30Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu
Phe 35 40 45Thr Val Pro Leu Gln Pro Pro Ala Ala Thr Ile Ser Gly Leu
Lys Pro 50 55 60Gly Val Asp Tyr Thr Ile Thr Gly Tyr Ala Val Thr Phe
His Arg Arg65 70 75 80Arg Gly Glu Leu Phe Thr Pro Ile Ser Ile Asn
Tyr Arg Thr 85 9015994PRTArtificial Sequencebinding polypeptide
159Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr
1 5 10 15Ser Leu Leu Ile Ser Trp Leu His Pro His Phe Pro Thr His
Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val
Gln Glu Phe 35 40 45Thr Val Pro Leu Gln Pro Pro Ala Ala Thr Ile Ser
Gly Leu Lys Pro 50 55 60Gly Val Asp Tyr Thr Ile Thr Gly Tyr Ala Val
Thr Leu Ile Arg Met65 70 75 80Asn Thr Glu Leu Phe Thr Pro Ile Ser
Ile Asn Tyr Arg Thr 85 9016094PRTArtificial Sequencebinding
polypeptide 160Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala
Thr Pro Thr 1 5 10 15Ser Leu Leu Ile Ser Trp Leu His Pro His Phe
Pro Thr His Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn
Ser Pro Val Gln Glu Phe 35 40 45Thr Val Pro Leu Gln Pro Pro Ala Ala
Thr Ile Ser Gly Leu Lys Pro 50 55 60Gly Val Asp Tyr Thr Ile Thr Gly
Tyr Ala Val Thr Cys Leu His Leu65 70 75 80Ile Thr Glu Leu Phe Thr
Pro Ile Ser Ile Asn Tyr Arg Thr 85 9016194PRTArtificial
Sequencebinding polypeptide 161Val Ser Asp Val Pro Arg Asp Leu Glu
Val Val Ala Ala Thr Pro Thr 1 5 10 15Ser Leu Leu Ile Ser Trp Leu
His Pro His Phe Pro Thr His Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 35 40 45Thr Val Pro Leu Gln
Pro Pro Ala Ala Thr Ile Ser Gly Leu Lys Pro 50 55 60Gly Val Asp Tyr
Thr Ile Thr Gly Tyr Ala Val Thr Val Leu Lys Leu65 70 75 80Thr Leu
Glu Leu Phe Thr Pro Ile Ser Ile
Asn Tyr Arg Thr 85 9016294PRTArtificial Sequencebinding polypeptide
162Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr
1 5 10 15Ser Leu Leu Ile Ser Trp Leu His Pro His Phe Pro Thr His
Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val
Gln Glu Phe 35 40 45Thr Val Pro Leu Gln Pro Pro Ala Ala Thr Ile Ser
Gly Leu Lys Pro 50 55 60Gly Val Asp Tyr Thr Ile Thr Gly Tyr Ala Val
Thr Val Leu Lys Leu65 70 75 80Thr Leu Glu Leu Phe Thr Pro Ile Ser
Ile Asn Tyr Arg Thr 85 9016394PRTArtificial Sequencebinding
polypeptide 163Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala
Thr Pro Thr 1 5 10 15Ser Leu Leu Ile Ser Trp Phe His Pro His Phe
Pro Thr His Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn
Ser Pro Val Gln Glu Phe 35 40 45Thr Val Pro Leu Gln Pro Pro Ala Ala
Thr Ile Ser Gly Leu Lys Pro 50 55 60Gly Val Asp Tyr Thr Ile Thr Gly
Tyr Ala Val Thr Val Leu Lys Leu65 70 75 80Thr Leu Glu Leu Phe Thr
Pro Ile Ser Ile Asn Tyr Arg Thr 85 9016494PRTArtificial
Sequencebinding polypeptide 164Val Ser Asp Val Pro Arg Asp Leu Glu
Val Val Ala Ala Thr Pro Thr 1 5 10 15Ser Leu Leu Ile Ser Trp Leu
His Pro His Phe Pro Thr His Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 35 40 45Thr Val Pro Leu Gln
Pro Pro Ala Ala Thr Ile Ser Gly Leu Lys Pro 50 55 60Gly Val Asp Tyr
Thr Ile Thr Gly Tyr Ala Val Thr Ala Leu Met Ala65 70 75 80Ser Gly
Glu Leu Phe Thr Pro Ile Ser Ile Asn Tyr Arg Thr 85
9016594PRTArtificial Sequencebinding polypeptide 165Val Ser Asp Val
Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr 1 5 10 15Ser Leu
Leu Ile Ser Trp Leu His Pro His Phe Pro Thr His Tyr Tyr 20 25 30Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 35 40
45Thr Val Pro Leu Gln Pro Pro Ala Ala Thr Ile Ser Gly Leu Lys Pro
50 55 60Gly Val Asp Tyr Thr Ile Thr Gly Tyr Ala Val Thr Ser Met Lys
Asn65 70 75 80Arg Leu Glu Leu Phe Thr Pro Ile Ser Ile Asn Tyr Arg
Thr 85 9016694PRTArtificial Sequencebinding polypeptide 166Val Ser
Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr 1 5 10
15Ser Leu Leu Ile Ser Trp Phe His Pro His Phe Pro Thr His Tyr Tyr
20 25 30Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu
Phe 35 40 45Thr Val Pro Leu Gln Pro Pro Ala Ala Thr Ile Ser Gly Leu
Lys Pro 50 55 60Gly Val Asp Tyr Thr Ile Thr Gly Tyr Ala Val Thr Leu
Arg Cys Leu65 70 75 80Ile Pro Glu Leu Phe Thr Pro Ile Ser Ile Asn
Tyr Arg Thr 85 9016794PRTArtificial Sequencebinding polypeptide
167Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr
1 5 10 15Ser Leu Leu Ile Ser Trp Leu His Pro His Phe Pro Thr His
Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val
Gln Glu Phe 35 40 45Thr Val Pro Leu Gln Pro Pro Ala Ala Thr Ile Ser
Gly Leu Lys Pro 50 55 60Gly Val Asp Tyr Thr Ile Thr Gly Tyr Ala Val
Thr Val Ser Arg Gln65 70 75 80Lys Ala Glu Leu Phe Thr Pro Ile Ser
Ile Asn Tyr Arg Thr 85 9016894PRTArtificial Sequencebinding
polypeptide 168Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala
Thr Pro Thr 1 5 10 15Ser Leu Leu Ile Ser Trp Arg His Pro His Phe
Pro Thr His Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn
Ser Pro Val Gln Glu Phe 35 40 45Thr Val Pro Leu Gln Pro Pro Ala Ala
Thr Ile Ser Gly Leu Lys Pro 50 55 60Gly Val Asp Tyr Thr Ile Thr Gly
Tyr Ala Val Thr Trp Ser Arg Thr65 70 75 80Gly Arg Glu Leu Phe Thr
Pro Ile Ser Ile Asn Tyr Arg Thr 85 9016994PRTArtificial
Sequencebinding polypeptide 169Val Ser Asp Val Pro Arg Asp Leu Glu
Val Val Ala Ala Thr Pro Thr 1 5 10 15Ser Leu Leu Ile Ser Trp Arg
His Pro His Phe Pro Thr His Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 35 40 45Thr Val Pro Leu Gln
Pro Pro Ala Ala Thr Ile Ser Gly Leu Lys Pro 50 55 60Gly Val Asp Tyr
Thr Ile Thr Gly Tyr Ala Val Thr Val Trp Arg Thr65 70 75 80Gly Arg
Glu Leu Phe Thr Pro Ile Ser Ile Asn Tyr Arg Thr 85
9017094PRTArtificial Sequencebinding polypeptide 170Val Ser Asp Val
Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr 1 5 10 15Ser Leu
Leu Ile Ser Trp Arg His Pro His Phe Pro Thr His Tyr Tyr 20 25 30Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 35 40
45Thr Val Pro Leu Gln Pro Pro Ala Ala Thr Ile Ser Gly Leu Lys Pro
50 55 60Gly Val Asp Tyr Thr Ile Thr Gly Tyr Ala Val Thr Thr Glu Arg
Thr65 70 75 80Gly Arg Glu Leu Phe Thr Pro Ile Ser Ile Asn Tyr Arg
Thr 85 9017194PRTArtificial Sequencebinding polypeptide 171Val Ser
Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr 1 5 10
15Ser Leu Leu Ile Ser Trp Arg His Pro His Phe Pro Thr His Tyr Tyr
20 25 30Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu
Phe 35 40 45Thr Val Pro Leu Gln Pro Pro Ala Ala Thr Ile Ser Gly Leu
Lys Pro 50 55 60Gly Val Asp Tyr Thr Ile Thr Gly Tyr Ala Val Thr Lys
Glu Arg Ser65 70 75 80Gly Arg Glu Leu Phe Thr Pro Ile Ser Ile Asn
Tyr Arg Thr 85 9017294PRTArtificial Sequencebinding polypeptide
172Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr
1 5 10 15Ser Leu Leu Ile Ser Trp Arg His Pro His Phe Pro Thr His
Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val
Gln Glu Phe 35 40 45Thr Val Pro Leu Gln Pro Pro Ala Ala Thr Ile Ser
Gly Leu Lys Pro 50 55 60Gly Val Asp Tyr Thr Ile Thr Gly Tyr Ala Val
Thr Leu Glu Arg Asn65 70 75 80Asp Arg Glu Leu Phe Thr Pro Ile Ser
Ile Asn Tyr Arg Thr 85 9017394PRTArtificial Sequencebinding
polypeptide 173Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala
Thr Pro Thr 1 5 10 15Ser Leu Leu Ile Ser Trp Arg His Pro His Phe
Pro Thr His Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn
Ser Pro Val Gln Glu Phe 35 40 45Thr Val Pro Leu Gln Pro Pro Ala Ala
Thr Ile Ser Gly Leu Lys Pro 50 55 60Gly Val Asp Tyr Thr Ile Thr Gly
Tyr Ala Val Thr Leu Glu Arg Asp65 70 75 80Gly Arg Glu Leu Phe Thr
Pro Ile Ser Ile Asn Tyr Arg Thr 85 9017494PRTArtificial
Sequencebinding polypeptide 174Val Ser Asp Val Pro Arg Asp Leu Glu
Val Val Ala Ala Thr Pro Thr 1 5 10 15Ser Leu Leu Ile Ser Trp Arg
His Pro His Phe Pro Thr Arg Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 35 40 45Thr Val Pro Leu Gln
Pro Pro Thr Ala Thr Ile Ser Gly Leu Lys Pro 50 55 60Gly Val Asp Tyr
Thr Ile Thr Gly Tyr Ala Val Thr Gln Gly Arg His65 70 75 80Lys Arg
Glu Leu Phe Thr Pro Ile Ser Ile Asn Tyr Arg Thr 85
9017594PRTArtificial Sequencebinding polypeptide 175Val Ser Asp Val
Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr 1 5 10 15Ser Leu
Leu Ile Ser Trp Arg His Pro His Phe Pro Thr Arg Tyr Tyr 20 25 30Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 35 40
45Thr Val Pro Leu Gln Pro Pro Leu Ala Thr Ile Ser Gly Leu Lys Pro
50 55 60Gly Val Asp Tyr Thr Ile Thr Gly Tyr Ala Val Thr Lys Glu Arg
Asn65 70 75 80Gly Arg Glu Leu Phe Thr Pro Ile Ser Ile Asn Tyr Arg
Thr 85 9017694PRTArtificial Sequencebinding polypeptide 176Val Ser
Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr 1 5 10
15Ser Leu Leu Ile Ser Trp Arg His Pro His Phe Pro Thr Arg Tyr Tyr
20 25 30Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu
Phe 35 40 45Thr Val Pro Leu Gln Pro Pro Ile Ala Thr Ile Ser Gly Leu
Lys Pro 50 55 60Gly Val Asp Tyr Thr Ile Thr Gly Tyr Ala Val Thr Met
Ala Gln Asn65 70 75 80Asp His Glu Leu Ile Thr Pro Ile Ser Ile Asn
Tyr Arg Thr 85 9017794PRTArtificial Sequencebinding polypeptide
177Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr
1 5 10 15Ser Leu Leu Ile Ser Trp Arg His Pro His Phe Pro Thr Arg
Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val
Gln Glu Phe 35 40 45Thr Val Pro Leu Gln Pro Pro Ile Ala Thr Ile Ser
Gly Leu Lys Pro 50 55 60Gly Val Asp Tyr Thr Ile Thr Gly Tyr Ala Val
Thr Met Ala Gln Asn65 70 75 80Asp His Glu Leu Ile Thr Pro Ile Ser
Ile Asn Tyr Arg Thr 85 9017894PRTArtificial Sequencebinding
polypeptide 178Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala
Thr Pro Thr 1 5 10 15Ser Leu Leu Ile Ser Trp Arg His Pro His Phe
Pro Thr Arg Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn
Ser Pro Val Gln Glu Phe 35 40 45Thr Val Pro Leu Gln Pro Thr Thr Ala
Thr Ile Ser Gly Leu Lys Pro 50 55 60Gly Val Asp Tyr Thr Ile Thr Gly
Tyr Ala Val Thr Trp Glu Arg Asn65 70 75 80Gly Arg Glu Leu Phe Thr
Pro Ile Ser Ile Asn Tyr Arg Thr 85 9017994PRTArtificial
Sequencebinding polypeptide 179Val Ser Asp Val Pro Arg Asp Leu Glu
Val Val Ala Ala Thr Pro Thr 1 5 10 15Ser Leu Leu Ile Ser Trp Arg
His Pro His Phe Pro Thr Arg Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 35 40 45Thr Val Pro Leu Gln
Pro Thr Val Ala Thr Ile Ser Gly Leu Lys Pro 50 55 60Gly Val Asp Tyr
Thr Ile Thr Gly Tyr Ala Val Thr Leu Glu Arg Asn65 70 75 80Asp Arg
Glu Leu Phe Thr Pro Ile Ser Ile Asn Tyr Arg Thr 85
9018094PRTArtificial Sequencebinding polypeptide 180Val Ser Asp Val
Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr 1 5 10 15Ser Leu
Leu Ile Ser Trp Arg Pro Pro His Phe Pro Thr Arg Tyr Tyr 20 25 30Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 35 40
45Thr Val Pro Leu Gln Pro Thr Val Ala Thr Ile Ser Gly Leu Lys Pro
50 55 60Gly Val Asp Tyr Thr Ile Thr Gly Tyr Ala Val Thr Leu Glu Arg
Asn65 70 75 80Asp Arg Glu Leu Phe Thr Pro Ile Ser Ile Asn Tyr Arg
Thr 85 9018194PRTArtificial Sequencebinding polypeptide 181Val Ser
Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr 1 5 10
15Ser Leu Leu Ile Ser Trp Arg His Pro His Phe Pro Thr Arg Tyr Tyr
20 25 30Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu
Phe 35 40 45Thr Val Pro Pro Gln Pro Pro Ala Ala Thr Ile Ser Gly Leu
Lys Pro 50 55 60Gly Val Asp Tyr Thr Ile Thr Gly Tyr Ala Val Thr Lys
Glu Arg Ser65 70 75 80Gly Arg Glu Leu Phe Thr Pro Ile Ser Ile Asn
Tyr Arg Thr 85 9018294PRTArtificial Sequencebinding polypeptide
182Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr
1 5 10 15Ser Leu Leu Ile Ser Trp Arg His Pro His Phe Pro Thr Arg
Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val
Gln Glu Phe 35 40 45Thr Val Pro Pro Gln Pro Pro Ala Ala Thr Ile Ser
Gly Leu Lys Pro 50 55 60Gly Val Asp Tyr Thr Ile Thr Gly Tyr Ala Val
Thr Leu Glu Arg Asn65 70 75 80Asp Arg Glu Leu Phe Thr Pro Ile Ser
Ile Asn Tyr Arg Thr 85 9018394PRTArtificial Sequencebinding
polypeptide 183Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala
Thr Pro Thr 1 5 10 15Ser Leu Leu Ile Ser Trp Cys His Pro His Phe
Pro Thr Arg Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn
Ser Pro Val Gln Glu Phe 35 40 45Thr Val Pro Leu Gln Pro Pro Ile Ala
Thr Ile Ser Gly Leu Lys Pro 50 55 60Gly Val Asp Tyr Thr Ile Thr Gly
Tyr Ala Val Thr Met Ala Gln Asn65 70 75 80Asp His Glu Leu Ile Thr
Pro Ile Ser Ile Asn Tyr Arg Thr 85 90184285DNAArtificial
Sequencebinding polypeptide 184atgggcgaag ttgttgctgc gacccccacc
agcctactga tcagctggcg ccacccgcac 60ttcccgacta gatattacag gatcacttac
ggagaaacag gaggaaatag ccctgtccag 120gagttcactg tgcctctgca
gccccccaca gctaccatca gcggccttaa acctggagtt 180gattatacca
tcactgtgta tgctgtcact gacggccgga acgggcgcct cctgagcatc
240ccaatttcca ttaattaccg cacagaaatt gacaaaccat gccag
285185264DNAArtificial Sequencebinding polypeptide 185atgggcgaag
ttgttgctgc gacccccacc agcctactga tcagctggcg ccacccgcac 60ttcccgacta
gatattacag gatcacttac ggagaaacag gaggaaatag ccctgtccag
120gagttcactg tgcctctgca gccccccaca gctaccatca gcggccttaa
acctggagtt 180gattatacca tcactgtgta tgctgtcact gacggccgga
acgggcgcct cctgagcatc 240ccaatttcca ttaattaccg caca
26418695PRTArtificial Sequencebinding polypeptide 186Met Gly Glu
Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp 1 5 10 15Arg
His Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu 20 25
30Thr Gly Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro
35 40 45Pro Thr Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr
Ile 50 55 60Thr Val Tyr Ala Val Thr Asp Gly Arg Asn Gly Arg Leu Leu
Ser Ile65 70 75 80Pro Ile Ser Ile Asn Tyr Arg Thr Glu Ile Asp Lys
Pro Ser Gln 85 90 9518795PRTArtificial Sequencebinding polypeptide
187Met Gly Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp
1 5 10 15Arg His Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr
Gly Glu 20 25 30Thr Gly Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro
Leu Gln Pro 35 40 45Pro Thr Ala Thr Ile Ser Gly Leu Lys Pro Gly Val
Asp Tyr Thr Ile 50 55 60Thr Val Tyr Ala Val Thr Asp Gly Arg Asn Gly
Arg Leu Leu Ser Ile65
70 75 80Pro Ile Ser Ile Asn Tyr Arg Thr Glu Ile Asp Lys Pro Cys Gln
85 90 95188102PRTArtificial Sequencebinding polypeptide 188Met Val
Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro 1 5 10
15Thr Ser Leu Leu Ile Ser Trp Arg His Pro His Phe Pro Thr Arg Tyr
20 25 30Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln
Glu 35 40 45Phe Thr Val Pro Leu Gln Pro Pro Thr Ala Thr Ile Ser Gly
Leu Lys 50 55 60Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr
Asp Gly Arg65 70 75 80Asn Gly Arg Leu Leu Ser Ile Pro Ile Ser Ile
Asn Tyr Arg Thr Glu 85 90 95Ile Asp Lys Pro Ser Gln
10018988PRTArtificial Sequencebinding polypeptide 189Met Gly Glu
Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp 1 5 10 15Arg
His Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu 20 25
30Thr Gly Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro
35 40 45Pro Thr Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr
Ile 50 55 60Thr Val Tyr Ala Val Thr Asp Gly Trp Asn Gly Arg Leu Leu
Ser Ile65 70 75 80Pro Ile Ser Ile Asn Tyr Arg Thr
8519088PRTArtificial Sequencebinding polypeptide 190Met Gly Glu Val
Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp 1 5 10 15Arg His
Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu 20 25 30Thr
Gly Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro 35 40
45Pro Thr Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile
50 55 60Thr Val Tyr Ala Val Thr Glu Gly Pro Asn Glu Arg Ser Leu Phe
Ile65 70 75 80Pro Ile Ser Ile Asn Tyr Arg Thr 8519195PRTArtificial
Sequencebinding polypeptide 191Met Val Ser Asp Val Pro Arg Asp Leu
Glu Val Val Ala Ala Thr Pro 1 5 10 15Thr Ser Leu Leu Ile Ser Trp
Arg His Pro His Phe Pro Thr Arg Tyr 20 25 30Tyr Arg Ile Thr Tyr Gly
Glu Thr Gly Gly Asn Ser Pro Val Gln Glu 35 40 45Phe Thr Val Pro Leu
Gln Pro Pro Thr Ala Thr Ile Ser Gly Leu Lys 50 55 60Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala Val Thr Glu Gly Pro65 70 75 80Asn Glu
Arg Ser Leu Phe Ile Pro Ile Ser Ile Asn Tyr Arg Thr 85 90
9519286PRTArtificial Sequencebinding polypeptide 192Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Val
50 55 60Tyr Ala Val Thr Asp Gly Arg Asn Gly Arg Leu Leu Ser Ile Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 8519394PRTArtificial
Sequencebinding polypeptide 193Val Ser Asp Val Pro Arg Asp Leu Glu
Val Val Ala Ala Thr Pro Thr 1 5 10 15Ser Leu Leu Ile Ser Trp Arg
His Pro His Phe Pro Thr Arg Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 35 40 45Thr Val Pro Leu Gln
Pro Pro Thr Ala Thr Ile Ser Gly Leu Lys Pro 50 55 60Gly Val Asp Tyr
Thr Ile Thr Val Tyr Ala Val Thr Asp Gly Arg Asn65 70 75 80Gly Arg
Leu Leu Ser Ile Pro Ile Ser Ile Asn Tyr Arg Thr 85
9019494PRTArtificial Sequencebinding polypeptide 194Gly Glu Val Val
Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg 1 5 10 15His Pro
His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr 20 25 30Gly
Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro 35 40
45Thr Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr
50 55 60Val Tyr Ala Val Thr Asp Gly Arg Asn Gly Arg Leu Leu Ser Ile
Pro65 70 75 80Ile Ser Ile Asn Tyr Arg Thr Glu Ile Asp Lys Pro Cys
Gln 85 9019594PRTArtificial Sequencebinding polypeptide 195Gly Glu
Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg 1 5 10
15His Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
20 25 30Gly Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro
Pro 35 40 45Thr Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr
Ile Thr 50 55 60Val Tyr Ala Val Thr Asp Gly Arg Asn Gly Arg Leu Leu
Ser Ile Pro65 70 75 80Ile Ser Ile Asn Tyr Arg Thr Glu Ile Asp Lys
Pro Ser Gln 85 9019686PRTArtificial Sequencebinding polypeptide
196Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His
1 5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln
Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr
Thr Ile Thr Val 50 55 60Tyr Ala Val Thr Glu Gly Pro Asn Glu Arg Ser
Leu Phe Ile Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
8519794PRTArtificial Sequencebinding polypeptide 197Val Ser Asp Val
Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr 1 5 10 15Ser Leu
Leu Ile Ser Trp Arg His Pro His Phe Pro Thr Arg Tyr Tyr 20 25 30Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 35 40
45Thr Val Pro Leu Gln Pro Pro Thr Ala Thr Ile Ser Gly Leu Lys Pro
50 55 60Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Glu Gly Pro
Asn65 70 75 80Glu Arg Ser Leu Phe Ile Pro Ile Ser Ile Asn Tyr Arg
Thr 85 9019810PRTArtificial Sequencebinding polypeptide 198Val Ala
Gln Asn Asp His Glu Leu Ile Thr 1 5 10199109PRTArtificial
Sequencebinding polypeptide 199Met Gly Val Ser Asp Val Pro Arg Asp
Leu Glu Val Val Ala Ala Thr 1 5 10 15Pro Thr Ser Leu Leu Ile Ser
Trp Arg His Pro His Phe Pro Thr Arg 20 25 30Tyr Tyr Arg Ile Thr Tyr
Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe Thr Val Pro
Leu Gln Pro Pro Leu Ala Thr Ile Ser Gly Leu 50 55 60Lys Pro Gly Val
Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Lys Glu65 70 75 80Arg Asn
Gly Arg Glu Leu Phe Thr Pro Ile Ser Ile Asn Tyr Arg Thr 85 90 95Glu
Ile Asp Lys Pro Cys Gln His His His His His His 100
10520087PRTArtificial Sequencebinding polypeptide 200Gly Glu Val
Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg 1 5 10 15His
Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr 20 25
30Gly Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro
35 40 45Thr Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile
Thr 50 55 60Val Tyr Ala Val Thr Asp Gly Trp Asn Gly Arg Leu Leu Ser
Ile Pro65 70 75 80Ile Ser Ile Asn Tyr Arg Thr 8520166DNAArtificial
Sequencebinding polypeptide 201gcgtaatacg actcactata gggacaatta
ctatttacaa ttacaatggt ttctgatgtt 60ccgagg 6620278DNAArtificial
Sequencebinding polypeptide 202gcgtaatacg actcactata gggacaatta
ctatttacaa ttacaatgga agttgttgct 60gcgaccccca ccagccta
7820357DNAArtificial Sequencebinding polypeptide 203tttttttttt
tttttttttt aaatagcgga tgccttgtcg tcgtcgtcct tgtagtc
5720469DNAArtificial Sequencebinding polypeptide 204atggtttctg
atgttccgag ggacctggaa gttgttgctg cgacccccac cagcctactg 60atcagctgg
6920566DNAArtificial Sequencebinding polypeptide 205aggcacagtg
aactcctgga cagggctatt tcctcctgtt tctccgtaag tgatcctgta 60atatct
6620666DNAArtificial Sequencebinding polypeptide 206agatattaca
ggatcactta cggagaaaca ggaggaaata gccctgtcca ggagttcact 60gtgcct
6620763DNAArtificial Sequencebinding polypeptide 207agtgacagca
tacacagtga tggtataatc aactccaggt ttaaggccgc tgatggtagc 60tgt
6320863DNAArtificial Sequencebinding polypeptide 208acagctacca
tcagcggcct taaacctgga gttgattata ccatcactgt gtatgctgtc 60act
6320977DNAArtificial Sequencebinding polypeptide 209tttttttttt
ttttttttta aatagcggat gccttgtcgt cgtcgtcctt gtagtctgtt 60cggtaattaa
tggaaat 7721058DNAArtificial Sequencebinding polypeptide
210ttttaaatag cggatgcctt gtcgtcgtcg tccttgtagt ctgttcggta attaatgg
5821130DNAArtificial Sequencebinding polypeptide 211gtgtatgctg
tcactatttc cattaattac 3021268DNAArtificial Sequencebinding
polypeptide 212taatacgact cactataggg acaattacta tttacaattc
tatcaataca atggtgtctg 60atgtgccg 6821372DNAArtificial
Sequencebinding polypeptide 213ccaggagatc agcagggagg tcggggtggc
agccaccact tccaggtcgc gcggcacatc 60agacaccatt gt
7221464DNAArtificial Sequencebinding polypeptide 214acctccctgc
tgatctcctg gcgccatccg cattttccga cccgctatta ccgcatcact 60tacg
6421563DNAArtificial Sequencebinding polypeptide 215cacagtgaac
tcctggaccg ggctattgcc tcctgtttcg ccgtaagtga tgcggtaata 60gcg
6321661DNAArtificial Sequencebinding polypeptide 216cggtccagga
gttcactgtg ccgctgcagc cgccggcggc taccatcagc ggccttaaac 60c
6121761DNAArtificial Sequencebinding polypeptide 217cggtccagga
gttcactgtg ccgnnsnnsn nsnnsnnsgc taccatcagc ggccttaaac 60c
6121859DNAArtificial Sequencebinding polypeptide 218agtgacagca
tacacagtga tggtataatc aacgccaggt ttaaggccgc tgatggtag
5921987DNAArtificial Sequencebinding polypeptide 219accatcactg
tgtatgctgt cactnnsnns nnsnnsnnsn nsgaactgtt taccccaatt 60tccatcaact
accgcacaga ctacaag 8722097DNAArtificial Sequencebinding polypeptide
220aaatagcgga tgcgcgtttg ttctgatctt ccttatttat gtgatgatgg
tggtgatgct 60tgtcgtcgtc gtccttgtag tctgtgcggt agttgat
9722151DNAArtificial Sequencebinding polypeptide 221tttttttttt
tttttttttt aaatagcgga tgcgcgtttg ttctgatctt c 5122221DNAArtificial
Sequencebinding polypeptide 222gcgcgtttgt tctgatcttc c
2122373DNAArtificial Sequencebinding polypeptide 223tgcctcctgt
ttcgccgtaa gtgatgcggt aatagcgsnn snnsnnsnns nnsnnsnncc 60agctgatcag
cag 7322469DNAArtificial Sequencebinding polypeptide 224gatggtagct
gtsnnsnnsn nsnnaggcac agtgaactcc tggacagggc tattgcctcc 60tgtttcgcc
6922568DNAArtificial Sequencebinding polypeptide 225gtgcggtaat
taatggaaat tggsnnsnns nnsnnsnnsn nsnnsnnsnn snnagtgaca 60gcatacac
6822621DNAArtificial Sequencebinding polypeptide 226cctcctgttt
ctccgtaagt g 2122721DNAArtificial Sequencebinding polypeptide
227cacttacgga gaaacaggag g 2122863DNAArtificial Sequencebinding
polypeptide 228acagctacca tcagcggcct taaacctggc gttgattata
ccatcactgt gtatgctgtc 60act 6322918DNAArtificial Sequencebinding
polypeptide 229agtgacagca tacacagt 1823075DNAArtificial
Sequencebinding polypeptide 230tttttttttt tttttttttt aaatagcgga
tgccttgtcg tcgtcgtcct tgtagtctgt 60gcggtaatta atgga
7523128DNAArtificial Sequencebinding polypeptide 231tagagaattc
atggagagca aggtgctg 2823239DNAArtificial Sequencebinding
polypeptide 232agggagagcg tcaggatgag ttccaagttc gtcttttcc
3923328DNAArtificial Sequencebinding polypeptide 233tagagaattc
atggagagca aggcgctg 2823439DNAArtificial Sequencebinding
polypeptide 234agggagagcg tcaggatgag ttccaagttg gtcttttcc
3923532DNAArtificial Sequencebinding polypeptide 235tagagaattc
atgatgtcgt cctggataag gt 3223639DNAArtificial Sequencebinding
polypeptide 236agggagagcg tcaggatgag atgttcccga ccggtttta
3923739DNAArtificial Sequencebinding polypeptide 237ggaaaagacg
aacttggaac tcatcctgac gctctccct 3923839DNAArtificial
Sequencebinding polypeptide 238ggaaaagacc aacttggaac tcatcctgac
gctctccct 3923931DNAArtificial Sequencebinding polypeptide
239tagactcgag tcaagagcaa gccacatagc t 3124039DNAArtificial
Sequencebinding polypeptide 240taaaaccggt cgggaacatc tcatcctgac
gctctccct 39241126PRTArtificial Sequencebinding polypeptide 241Glu
Ile Val Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10
15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn
20 25 30Ser Val Ala Trp Tyr Arg Lys Lys Pro Gly Gln Ala Pro Arg Leu
Leu 35 40 45Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg
Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Arg Leu Glu65 70 75 80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Ala Thr
Ile Val Ala Gly Asp 85 90 95His Tyr Phe Asp His Trp Gly Gln Gly Thr
Lys Val Glu Ile Lys Ala 100 105 110Asp Gly Ser Asp Tyr Lys Asp Asp
Asp Asp Lys Ala Ser Ala 115 120 125242126PRTArtificial
Sequencebinding polypeptide 242Glu Ile Val Met Thr Gln Ser Pro Gly
Thr Leu Ser Leu Ser Pro Gly 1 5 10 15Glu Arg Ala Thr Leu Ser Cys
Arg Ala Ser Gln Ser Val Ser Ser Asn 20 25 30Ser Val Ala Trp Tyr Arg
Lys Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45Ile Tyr Gly Ala Ser
Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60Gly Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu65 70 75 80Pro Glu
Asp Phe Ala Val Tyr Tyr Cys Ala Thr Ile Val Ala Gly Asp 85 90 95His
Tyr Leu Asp His Trp Gly Gln Gly Thr Lys Val Glu Ile Lys Ala 100 105
110Asp Gly Ser Asp Tyr Lys Asp Asp Asp Asp Lys Ala Ser Ala 115 120
125243126PRTArtificial Sequencebinding polypeptide 243Glu Ile Val
Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15Glu
Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn 20 25
30Ser Val Ala Trp Tyr Arg Lys Lys Pro Gly Gln Ala Pro Arg Leu Leu
35 40 45Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe
Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg
Leu Glu65 70 75 80Pro Glu Asp Phe Ala
Val Tyr Tyr Cys Ala Thr Ile Val Ala Gly Asp 85 90 95His Tyr Leu Asp
His Trp Gly Gln Gly Thr Lys Val Glu Ile Lys Ala 100 105 110Asp Gly
Ser Asp Tyr Lys Asp Asp Asp Asp Lys Ala Ser Ala 115 120
125244126PRTArtificial Sequencebinding polypeptide 244Glu Ile Val
Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15Glu
Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn 20 25
30Ser Val Ala Trp Tyr Arg Lys Lys Pro Gly Gln Ala Pro Arg Leu Leu
35 40 45Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe
Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg
Leu Glu65 70 75 80Pro Glu Asp Phe Ala Ala Tyr Tyr Cys Ala Thr Ile
Val Ala Gly Asp 85 90 95His Tyr Phe Asp His Trp Gly Gln Gly Thr Lys
Val Glu Ile Ile Ala 100 105 110Asp Gly Ser Asp Tyr Lys Asp Asp Asp
Asp Lys Ala Ser Ala 115 120 125245126PRTArtificial Sequencebinding
polypeptide 245Glu Ile Val Met Thr Gln Ser Pro Gly Thr Leu Ser Leu
Ser Pro Gly 1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln
Ser Val Ser Ser Asn 20 25 30Ser Val Ala Trp Tyr Arg Lys Lys Pro Gly
Gln Ala Pro Arg Leu Leu 35 40 45Ile Tyr Gly Ala Ser Ser Arg Ala Thr
Gly Ile Pro Asp Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Arg Leu Glu65 70 75 80Pro Glu Asp Phe Ala Val
Tyr Tyr Cys Ala Thr Ile Val Ala Gly Asp 85 90 95His Tyr Phe Asp His
Trp Gly Gln Gly Thr Lys Val Glu Ile Lys Ala 100 105 110Asp Gly Ser
Asp Tyr Lys Asp Asp Asp Asp Lys Ala Ser Ala 115 120
125246126PRTArtificial Sequencebinding polypeptide 246Glu Ile Val
Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15Glu
Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn 20 25
30Ser Val Ala Trp Tyr Arg Lys Lys Pro Gly Gln Ala Pro Arg Leu Leu
35 40 45Ile Tyr Gly Ala Ser Ser Arg Ala Ala Gly Ile Pro Asp Arg Phe
Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg
Leu Glu65 70 75 80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Ala Thr Ile
Val Ala Gly Asp 85 90 95His Tyr Phe Asp His Trp Gly Gln Gly Thr Lys
Val Glu Ile Lys Ala 100 105 110Asp Gly Ser Asp Tyr Lys Asp Asp Asp
Asp Lys Ala Ser Ala 115 120 125247126PRTArtificial Sequencebinding
polypeptide 247Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu
Ser Pro Gly 1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln
Ser Val Gly Ser Asn 20 25 30Ser Val Ala Trp Tyr Arg Lys Lys Pro Gly
Gln Ala Pro Arg Leu Leu 35 40 45Ile Tyr Gly Ala Ser Ser Arg Ala Thr
Gly Ile Pro Asp Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Arg Leu Glu65 70 75 80Pro Glu Asp Phe Ala Val
Tyr Tyr Cys Ala Thr Ile Val Ala Gly Asp 85 90 95His Tyr Phe Asp His
Trp Gly Gln Gly Thr Lys Val Glu Ile Lys Ala 100 105 110Asp Gly Ser
Asp Tyr Lys Asp Asp Asp Asp Lys Ala Ser Ala 115 120
125248126PRTArtificial Sequencebinding polypeptide 248Ala Ile Gln
Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15Glu
Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn 20 25
30Ser Val Ala Trp Tyr Arg Lys Lys Pro Gly Gln Ala Pro Arg Leu Leu
35 40 45Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe
Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg
Leu Glu65 70 75 80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Ala Thr Ile
Val Ala Gly Asp 85 90 95His Tyr Phe Asp His Trp Gly Gln Gly Thr Lys
Val Glu Ile Lys Ala 100 105 110Asp Gly Ser Asp Tyr Lys Asp Asp Asp
Asp Lys Ala Ser Ala 115 120 125249126PRTArtificial Sequencebinding
polypeptide 249Ala Ile Gln Met Thr Gln Ser Pro Gly Thr Leu Ser Leu
Ser Pro Gly 1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln
Ser Val Ser Ser Asn 20 25 30Ser Val Ala Trp Tyr Arg Lys Lys Pro Gly
Gln Ala Pro Arg Leu Leu 35 40 45Ile Tyr Gly Ala Ser Ser Arg Ala Thr
Gly Ile Pro Asp Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Arg Leu Glu65 70 75 80Pro Glu Asp Phe Ala Val
Tyr Tyr Cys Ala Thr Ile Val Ala Gly Asp 85 90 95His Tyr Phe Asp His
Trp Gly Gln Gly Thr Lys Val Glu Ile Lys Ala 100 105 110Asp Gly Ser
Asp Tyr Lys Asp Asp Asp Asp Lys Ala Ser Ala 115 120
125250126PRTArtificial Sequencebinding polypeptide 250Glu Ile Val
Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15Glu
Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn 20 25
30Ser Val Ala Trp Tyr Arg Lys Lys Pro Gly Gln Ala Pro Arg Leu Leu
35 40 45Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe
Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg
Leu Glu65 70 75 80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Ala Thr Ile
Val Ala Gly Asp 85 90 95His Tyr Phe Asp His Trp Gly Gln Gly Thr Lys
Val Glu Ile Lys Ala 100 105 110Asp Gly Ser Asp Tyr Lys Asp Asp Asp
Asp Lys Ala Ser Ala 115 120 125251126PRTArtificial Sequencebinding
polypeptide 251Glu Ile Val Met Thr Gln Ser Pro Gly Thr Leu Ser Leu
Ser Pro Gly 1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln
Ser Val Ser Ser Asn 20 25 30Ser Val Ala Trp Tyr Arg Lys Lys Pro Gly
Gln Ala Pro Arg Leu Leu 35 40 45Ile Tyr Gly Ala Ser Ser Arg Ala Thr
Gly Ile Pro Asp Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Arg Leu Glu65 70 75 80Pro Glu Asp Phe Ala Val
Tyr Tyr Cys Ala Thr Ile Val Ala Gly Asp 85 90 95His Tyr Phe Asp His
Trp Gly Gln Gly Thr Lys Val Glu Ile Lys Ala 100 105 110Asp Gly Ser
Asp Tyr Lys Asp Asp Asp Asp Lys Ala Ser Ala 115 120
125252126PRTArtificial Sequencebinding polypeptide 252Glu Ile Val
Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15Glu
Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn 20 25
30Ser Val Ala Trp Tyr Arg Lys Lys Pro Gly Gln Ala Pro Arg Leu Leu
35 40 45Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe
Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg
Leu Glu65 70 75 80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Ala Thr Ile
Val Ala Gly Asp 85 90 95His Tyr Phe Asp His Trp Gly Gln Gly Thr Lys
Val Glu Ile Lys Ala 100 105 110Asp Gly Ser Asp Tyr Lys Asp Asp Asp
Asp Lys Ala Ser Ala 115 120 125253126PRTArtificial Sequencebinding
polypeptide 253Glu Ile Val Met Thr Gln Ser Pro Gly Thr Leu Ser Leu
Ser Pro Gly 1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln
Ser Val Ser Ser Asn 20 25 30Ser Val Ala Trp Tyr Arg Lys Lys Pro Gly
Gln Ala Pro Arg Leu Leu 35 40 45Ile Tyr Gly Ala Ser Ser Arg Ala Thr
Gly Ile Pro Asp Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Arg Leu Glu65 70 75 80Pro Glu Asp Phe Ala Val
Tyr Tyr Cys Ala Thr Ile Val Ala Gly Asp 85 90 95His Tyr Phe Asp His
Trp Gly Gln Gly Thr Lys Val Glu Ile Lys Ala 100 105 110Asp Gly Ser
Asp Tyr Lys Asp Asp Asp Asp Lys Ala Ser Ala 115 120
125254126PRTArtificial Sequencebinding polypeptide 254Glu Ile Val
Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15Glu
Arg Ala Thr Leu Ser Cys Arg Thr Ser Gln Ser Val Ser Ser Asn 20 25
30Ser Val Ala Trp Tyr Arg Lys Lys Pro Gly Gln Ala Pro Arg Leu Leu
35 40 45Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe
Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg
Leu Glu65 70 75 80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Ala Thr Ile
Val Ala Gly Asp 85 90 95His Tyr Phe Asp His Trp Gly Gln Gly Thr Lys
Val Glu Ile Lys Ala 100 105 110Asp Gly Ser Asp Tyr Lys Asp Asp Asp
Asp Lys Ala Ser Ala 115 120 125255126PRTArtificial Sequencebinding
polypeptide 255Glu Ile Val Met Thr Gln Ser Pro Asp Thr Leu Ser Leu
Ser Pro Gly 1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln
Ser Val Ser Ser Asn 20 25 30Ser Val Ala Trp Tyr Arg Lys Lys Pro Gly
Gln Ala Pro Arg Leu Leu 35 40 45Ile Tyr Gly Ala Ser Ser Arg Ala Thr
Gly Ile Pro Asp Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Arg Leu Glu65 70 75 80Pro Glu Asp Phe Ala Val
Tyr Tyr Cys Ala Thr Ile Val Ala Gly Asp 85 90 95His Tyr Phe Asp His
Trp Gly Gln Gly Thr Lys Val Glu Ile Lys Ala 100 105 110Asp Gly Ser
Asp Tyr Lys Asp Asp Asp Asp Lys Ala Ser Ala 115 120
125256126PRTArtificial Sequencebinding polypeptide 256Glu Ile Val
Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15Gly
Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn 20 25
30Ser Val Ala Trp Tyr Arg Lys Lys Pro Gly Gln Ala Pro Arg Leu Leu
35 40 45Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe
Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg
Leu Glu65 70 75 80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Ala Thr Ile
Val Ala Gly Asp 85 90 95His Tyr Phe Asp His Trp Gly Gln Gly Thr Lys
Val Glu Ile Lys Ala 100 105 110Asp Gly Ser Asp Tyr Lys Asp Asp Asp
Asp Lys Ala Ser Ala 115 120 125257125PRTArtificial Sequencebinding
polypeptide 257Glu Ile Val Met Thr Gln Ser Pro Gly Thr Leu Ser Leu
Ser Pro Gly 1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln
Ser Val Ser Ser Ser 20 25 30Ser Leu Ala Trp Tyr Arg Gln Lys Pro Gly
Gln Ala Pro Arg Leu Leu 35 40 45Ile Tyr Gly Ala Ser Asn Arg Ala Thr
Gly Ile Pro Asp Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Ser Leu Gln65 70 75 80Pro Glu Asp Phe Ala Val
Tyr Tyr Cys Ala Lys Leu Thr Tyr Tyr Gly 85 90 95Ser Gly Arg Asn Trp
Gly Gln Gly Thr Lys Leu Glu Ile Lys Ala Asp 100 105 110Gly Ser Asp
Tyr Lys Asp Asp Asp Asp Lys Ala Ser Ala 115 120
125258125PRTArtificial Sequencebinding polypeptide 258Glu Ile Val
Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15Glu
Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25
30Ser Leu Ala Trp Tyr Arg Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu
35 40 45Ile Tyr Gly Ala Ser Asn Arg Ala Thr Gly Ile Pro Asp Arg Phe
Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln65 70 75 80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Ala Lys Leu
Thr Tyr Tyr Gly 85 90 95Ser Gly Arg Asn Trp Gly Gln Gly Thr Lys Leu
Glu Ile Lys Ala Asp 100 105 110Gly Ser Asp Tyr Lys Asp Asp Asp Asp
Lys Ala Ser Ala 115 120 125259125PRTArtificial Sequencebinding
polypeptide 259Glu Ile Val Met Thr Gln Ser Pro Gly Thr Leu Ser Leu
Ser Pro Gly 1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln
Ser Val Ser Ser Ser 20 25 30Ser Leu Ala Trp Tyr Arg Gln Lys Pro Gly
Gln Ala Pro Arg Leu Leu 35 40 45Ile Tyr Gly Ala Ser Asn Arg Ala Thr
Gly Ile Pro Asp Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Ser Leu Gln65 70 75 80Pro Glu Asp Phe Ala Val
Tyr Tyr Cys Ala Lys Leu Thr Tyr Tyr Gly 85 90 95Ser Gly Arg Asn Trp
Gly Gln Gly Thr Lys Leu Glu Ile Lys Ala Asp 100 105 110Gly Ser Asp
Tyr Lys Asp Asp Asp Asp Lys Ala Ser Ala 115 120
125260125PRTArtificial Sequencebinding polypeptide 260Glu Ile Val
Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15Glu
Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25
30Ser Leu Ala Trp Tyr Arg Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu
35 40 45Ile Tyr Gly Ala Ser Asn Arg Ala Thr Gly Ile Pro Asp Arg Phe
Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln65 70 75 80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Ala Lys Leu
Thr Tyr Tyr Gly 85 90 95Ser Gly Arg Asn Trp Gly Gln Gly Thr Lys Leu
Glu Ile Lys Ala Asp 100 105 110Gly Ser Asp Tyr Lys Asp Asp Asp Asp
Lys Ala Ser Ala 115 120 125261125PRTArtificial Sequencebinding
polypeptide 261Glu Ile Val Met Thr Gln Ser Pro Gly Thr Leu Ser Leu
Ser Pro Gly 1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln
Ser Val Ser Ser Ser 20 25 30Ser Leu Ala Trp Tyr Arg Gln Lys Pro Gly
Gln Ala Pro Arg Leu Leu 35 40 45Ile Tyr Gly Ala Ser Asn Arg Ala Thr
Gly Ile Pro Asp Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Ser Leu Gln65 70 75 80Pro Glu Asp Phe Ala Val
Tyr Tyr Cys Ala Lys Leu Thr Tyr Tyr Gly 85 90 95Ser Gly Arg Asn Trp
Gly Gln Gly Thr Lys Leu Glu Ile Ile Ala Asp 100 105 110Gly Ser Asp
Tyr Lys Asp Asp Asp Asp Lys Ala Ser Ala 115 120
125262125PRTArtificial Sequencebinding polypeptide 262Glu Ile Val
Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15Asp
Arg Ala Thr Leu Ser Cys Arg Thr Ser Gln Ser Val Ser Ser Ser 20
25 30Ser Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu
Leu 35 40 45Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg
Phe Ser 50 55 60Gly Ser Gly Pro Gly Thr Asp Phe Thr Leu Thr Val Ser
Arg Leu Glu65 70 75 80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Ala Lys
Leu Thr Tyr Tyr Gly 85 90 95Ser Gly Arg Asn Trp Gly Gln Gly Thr Lys
Val Glu Ile Lys Ala Asp 100 105 110Gly Ser Asp Tyr Lys Asp Asp Asp
Asp Lys Ala Ser Ala 115 120 125263121PRTArtificial Sequencebinding
polypeptide 263Glu Ile Val Met Thr Gln Ser Pro Gly Thr Leu Ser Val
Ser Pro Gly 1 5 10 15Gly Arg Ala Ser Leu Pro Cys Arg Ala Ser Gln
Thr Val Thr Asn Asn 20 25 30Tyr Leu Ala Trp Tyr Gln Gln Lys Ser Gly
Gln Ala Pro Arg Leu Leu 35 40 45Ile Phe Gly Ala Ser Asn Arg Ala Thr
Gly Ile Pro Glu Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Ile Ile Ser Arg Leu Glu65 70 75 80Ala Glu Asp Phe Ala Val
Tyr Tyr Cys Val Ser Leu Lys Gly Arg Asp 85 90 95Trp Gly Gln Gly Thr
Lys Leu Glu Ile Lys Ala Asp Gly Ser Asp Tyr 100 105 110Lys Asp Asp
Asp Asp Lys Ala Ser Ala 115 120264126PRTArtificial Sequencebinding
polypeptide 264Glu Ile Gly Met Thr Gln Ser Pro Ala Thr Leu Ser Val
Ser Pro Gly 1 5 10 15Lys Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln
Ser Val Gly Ile Lys 20 25 30Leu Ala Gly Tyr Gln Gln Lys Leu Gly Gln
Ala Pro Arg Leu Leu Ile 35 40 45Tyr Glu Ala Ser Thr Arg Ala Thr Gly
Val Pro Thr Arg Phe Asn Gly 50 55 60Ser Gly Ser Gly Thr Glu Phe Thr
Leu Thr Ile Asn Ser Leu Gln Ser65 70 75 80Glu Asp Phe Ala Val Tyr
Tyr Cys Thr Gly Gly Thr Gly Asn Tyr Tyr 85 90 95Gly Trp Phe Asp Pro
Trp Gly Gln Gly Thr Lys Leu Glu Ile Lys Ala 100 105 110Tyr Gly Ser
Asp Tyr Glu Asp Asp Asp Asp Xaa Ala Ser Ala 115 120
125265123PRTArtificial Sequencebinding polypeptide 265Glu Ile Val
Leu Thr Gln Ser Pro Asp Phe Gln Ser Val Thr Pro Lys 1 5 10 15Lys
Lys Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Val Thr Ser 20 25
30Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Ser Leu Leu Ile
35 40 45Tyr Gly Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser
Gly 50 55 60Cys Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Gly Leu
Gln Pro65 70 75 80Glu Asp Ala Ala Val Tyr Tyr Cys Ala Gly Asp Phe
Gly Gly Gln Leu 85 90 95Pro Tyr Trp Gly Gln Gly Thr Lys Leu Glu Ile
Lys Ala Asp Gly Ser 100 105 110Asp Tyr Lys Asp Asp Asp Asp Lys Ala
Ser Ala 115 120266128PRTArtificial Sequencebinding polypeptide
266Gln Pro Val Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln
1 5 10 15Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly
Gly Tyr 20 25 30Asn Tyr Val Phe Trp Tyr Gln Gln His Pro Gly Lys Ala
Pro Lys Val 35 40 45Met Ile Tyr Glu Asp Thr Glu Arg Pro Ser Gly Val
Ser Asn Arg Phe 50 55 60Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu
Thr Ile Ser Gly Leu65 70 75 80Gln Ala Glu Asp Glu Ala Val Tyr Tyr
Cys Ala Arg Arg Asn Tyr Gly 85 90 95Ser Gly Ser Trp Ile Asp Tyr Trp
Gly Gln Gly Thr Lys Leu Thr Val 100 105 110Leu Ala Asp Gly Ser Asp
Tyr Lys Asp Asp Asp Asp Lys Ala Ser Ala 115 120
125267126PRTArtificial Sequencebinding polypeptide 267Ala Ile Gln
Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15Glu
Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Arg Thr Lys 20 25
30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Arg Leu Leu Ile
35 40 45Tyr Asp Ala Ser Thr Arg Ala Thr Gly Val Pro Ala Arg Phe Ser
Gly 50 55 60Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu
Gln Ser65 70 75 80Glu Asp Phe Ala Val Tyr Tyr Cys Thr Gly Gly Thr
Gly Asn Tyr Tyr 85 90 95Gly Trp Phe Asp Pro Trp Gly Gln Gly Thr Lys
Val Arg Ser Lys Gln 100 105 110Met Ala Ala Thr Thr Arg Thr Thr Thr
Thr Arg His Pro Leu 115 120 125268127PRTArtificial Sequencebinding
polypeptide 268Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala
Pro Gly Gln 1 5 10 15Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser
Asn Ile Gly Ala Gly 20 25 30Tyr Asp Val His Trp Tyr Gln Gln Leu Pro
Gly Thr Ala Pro Lys Leu 35 40 45Leu Ile Tyr Gly Asn Ser Asn Arg Pro
Ser Gly Val Pro Asp Arg Phe 50 55 60Ser Gly Ser Lys Ser Gly Thr Ser
Ala Ser Leu Ala Ile Thr Gly Leu65 70 75 80Gln Ala Glu Asp Glu Ala
Val Tyr Tyr Cys Ala Ser Ala Ile Pro Gly 85 90 95Thr Arg Ile Leu Arg
Asp Trp Gly Gln Gly Thr Lys Leu Thr Val Leu 100 105 110Ala Asp Gly
Ser Asp Tyr Arg Thr Thr Thr Thr Arg His Pro Leu 115 120
125269123PRTArtificial Sequencebinding polypeptide 269Glu Ile Val
Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15Glu
Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn 20 25
30Leu Ala Trp Tyr Gln Gln Arg Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45Tyr Gly Ala Ser Ile Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser
Gly 50 55 60Ser Glu Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu
Glu Pro65 70 75 80Glu Asp Phe Ala Val Tyr Tyr Cys Ala Thr Glu Phe
Arg Gly Tyr Ala 85 90 95Gly Tyr Trp Gly Gln Gly Thr Lys Leu Glu Ile
Lys Ala Asp Gly Ser 100 105 110Asp Tyr Lys Asp Asp Asp Asp Lys Ala
Ser Ala 115 120270125PRTArtificial Sequencebinding polypeptide
270Glu Ile Val Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly
1 5 10 15Arg Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser
Ser Ser 20 25 30Ser Leu Ala Trp Tyr Arg Gln Lys Pro Gly Gln Ala Pro
Arg Leu Leu 35 40 45Ile Tyr Gly Ala Ser Asn Arg Ala Thr Gly Ile Pro
Asp Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln65 70 75 80Pro Glu Asp Phe Ala Val Tyr Tyr Cys
Ala Lys Leu Thr Tyr Tyr Gly 85 90 95Ser Gly Arg Asn Trp Gly Gln Gly
Thr Lys Leu Glu Ile Lys Ala Asp 100 105 110Gly Ser Asp Tyr Lys Asp
Asp Asp Asp Lys Ala Ser Ala 115 120 125271125PRTArtificial
Sequencebinding polypeptide 271Gly Ile Val Leu Thr Gln Ser Pro Gly
Thr Leu Ser Leu Ser Pro Gly 1 5 10 15Glu Arg Ala Thr Leu Ser Cys
Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30Ser Leu Ala Trp Tyr Arg
Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45Ile Tyr Gly Ala Ser
Asn Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60Gly Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln65 70 75 80Pro Glu
Asp Phe Ala Val Tyr Tyr Cys Ala Lys Leu Thr Tyr Tyr Gly 85 90 95Ser
Gly Arg Asn Trp Gly Gln Gly Thr Lys Leu Glu Ile Lys Ala Asp 100 105
110Gly Ser Asp Tyr Lys Asp Asp Asp Asp Lys Ala Ser Ala 115 120
125272125PRTArtificial Sequencebinding polypeptide 272Glu Ile Val
Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15Glu
Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Ser Ser 20 25
30Ser Leu Ala Trp Tyr Arg Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu
35 40 45Ile Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Asp Arg Phe
Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Ser Leu Thr Ile Ser Ser
Leu Gln65 70 75 80Pro Glu Asp Ala Ala Val Tyr Tyr Cys Ala Lys Leu
Thr Tyr Tyr Gly 85 90 95Ser Gly Arg Asn Trp Gly Gln Gly Thr Lys Leu
Glu Ile Lys Ala Asp 100 105 110Gly Ser Asp Tyr Lys Asp Asp Asp Asp
Lys Ala Ser Ala 115 120 125273126PRTArtificial Sequencebinding
polypeptide 273Glu Ile Val Met Thr Gln Ser Pro Gly Ala Leu Ser Leu
Ser Pro Gly 1 5 10 15Glu Gly Ala Thr Leu Ser Cys Arg Ala Ser Gln
Ser Val Ser Ser Asn 20 25 30Ser Val Ala Trp Tyr Arg Lys Lys Pro Gly
Gln Ala Pro Arg Leu Leu 35 40 45Ile Tyr Gly Ala Ser Ser Arg Ala Thr
Gly Ile Pro Asp Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Arg Leu Glu65 70 75 80Pro Glu Asp Phe Ala Val
Tyr Tyr Cys Ala Thr Ile Val Ala Gly Asp 85 90 95His Tyr Phe Asp His
Trp Gly Gln Gly Thr Lys Val Glu Ile Lys Glu 100 105 110Asp Gly Ser
Asp Tyr Lys Asp Asp Asp Asp Lys Ala Ser Ala 115 120
125274126PRTArtificial Sequencebinding polypeptide 274Glu Ile Val
Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15Glu
Gly Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn 20 25
30Ser Val Ala Trp Tyr Arg Lys Lys Pro Gly Gln Ala Pro Arg Leu Leu
35 40 45Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe
Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg
Leu Glu65 70 75 80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Ala Thr Ile
Val Ala Gly Asp 85 90 95His Tyr Phe Asp His Trp Gly Gln Gly Thr Lys
Val Glu Ile Lys Ala 100 105 110Asp Gly Ser Asp Tyr Lys Asp Asp Asp
Asp Lys Ala Ser Ala 115 120 125275126PRTArtificial Sequencebinding
polypeptide 275Glu Ile Val Met Thr Gln Ser Pro Gly Thr Leu Ser Leu
Ser Pro Gly 1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln
Ser Val Ser Ser Asn 20 25 30Ser Val Ala Trp Tyr Arg Lys Lys Pro Gly
Leu Ala Pro Arg Leu Leu 35 40 45Ile Tyr Gly Ala Ser Ser Arg Ala Thr
Gly Ile Pro Asp Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Arg Leu Glu65 70 75 80Pro Glu Asp Phe Ala Val
Tyr Tyr Cys Ala Thr Ile Val Ala Gly Asp 85 90 95His Tyr Phe Asp His
Trp Gly Gln Gly Thr Lys Val Glu Ile Lys Ala 100 105 110Asp Gly Ser
Asp Tyr Lys Asp Asp Asp Asp Lys Ala Ser Ala 115 120
125276126PRTArtificial Sequencebinding polypeptide 276Glu Ile Val
Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15Glu
Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn 20 25
30Ser Val Ala Trp Tyr Arg Lys Lys Pro Gly Gln Ala Pro Arg Leu Leu
35 40 45Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe
Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg
Leu Glu65 70 75 80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Ala Thr Ile
Val Ala Gly Asp 85 90 95His Tyr Phe Asp His Trp Gly Gln Gly Thr Lys
Val Glu Ile Lys Ala 100 105 110Asp Gly Ser Asp Tyr Lys Asp Asp Asp
Asp Lys Ala Ser Ala 115 120 125277126PRTArtificial Sequencebinding
polypeptide 277Glu Ile Val Met Thr Gln Ser Pro Gly Thr Leu Ser Leu
Ser Pro Gly 1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln
Ser Val Ser Ser Asn 20 25 30Ser Val Ala Trp Tyr Arg Lys Lys Pro Gly
Gln Ala Pro Arg Leu Leu 35 40 45Ile Tyr Gly Ala Ser Ser Arg Ala Thr
Ser Ile Pro Asp Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Arg Leu Glu65 70 75 80Pro Glu Asp Phe Ala Val
Tyr Tyr Cys Ala Thr Ile Val Ala Gly Asp 85 90 95His Tyr Phe Asp His
Trp Gly Gln Gly Thr Lys Val Glu Ile Ile Ala 100 105 110Asp Gly Ser
Asp Tyr Lys Asp Asp Asp Asp Lys Ala Ser Ala 115 120
125278126PRTArtificial Sequencebinding polypeptide 278Ala Ile Gln
Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15Gly
Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn 20 25
30Ser Val Ala Trp Tyr Arg Lys Lys Pro Gly Gln Ala Pro Arg Leu Leu
35 40 45Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe
Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg
Leu Glu65 70 75 80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Ala Thr Ile
Val Ala Gly Asp 85 90 95His Tyr Phe Asp His Trp Gly Gln Gly Thr Lys
Val Glu Ile Lys Ala 100 105 110Asp Gly Ser Asp Tyr Lys Asp Asp Asp
Asp Lys Ala Ser Ala 115 120 125279126PRTArtificial Sequencebinding
polypeptide 279Lys Ile Val Met Thr Gln Ser Pro Gly Thr Leu Ser Leu
Ser Pro Gly 1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln
Ser Val Ser Ser Asn 20 25 30Ser Val Ala Trp Tyr Arg Lys Lys Pro Gly
Gln Ala Pro Arg Leu Leu 35 40 45Ile Tyr Gly Ala Ser Ser Arg Ala Thr
Gly Ile Pro Asp Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Arg Leu Glu65 70 75 80Pro Glu Asp Phe Ala Val
Tyr Tyr Cys Ala Thr Ile Val Ala Gly Asp 85 90 95His Tyr Phe Asp His
Trp Gly Gln Gly Thr Lys Val Glu Ile Lys Ala 100 105 110Asp Gly Ser
Asp Tyr Lys Asp Asp Asp Asp Lys Ala Ser Ala 115 120
125280126PRTArtificial Sequencebinding polypeptide 280Asp Ile Val
Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15Glu
Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn 20 25
30Ser Val Ala Trp Tyr Arg Lys Lys Pro Gly Gln Ala Pro Arg Leu Leu
35 40 45Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe
Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg
Leu Glu65 70 75 80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Ala Thr Ile
Val Ala Gly Asp 85 90 95His Tyr Phe Asp His Trp Gly Gln Gly Thr Lys
Val Glu Ile Lys Ala 100 105 110Asp Gly Ser Asp Tyr Lys Asp Asp Asp
Asp Lys Ala Ser Ala 115
120 125281126PRTArtificial Sequencebinding polypeptide 281Glu Ile
Val Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10
15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn
20 25 30Ser Val Ala Trp Tyr Arg Lys Lys Pro Gly Gln Ala Pro Arg Leu
Leu 35 40 45Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg
Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Arg Leu Glu65 70 75 80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Ala Thr
Ile Val Ala Gly Asp 85 90 95His Tyr Phe Asp His Trp Gly Gln Gly Thr
Lys Val Glu Ile Lys Ala 100 105 110Asp Gly Ser Asp Tyr Lys Asp Asp
Asp Asp Lys Ala Ser Ala 115 120 125282126PRTArtificial
Sequencebinding polypeptide 282Glu Ile Val Met Thr Gln Ser Pro Gly
Thr Leu Ser Leu Ser Pro Gly 1 5 10 15Glu Arg Ala Thr Leu Ser Cys
Arg Ala Ser Gln Ser Val Ser Ser Asn 20 25 30Ser Val Ala Trp Tyr Arg
Lys Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45Ile Tyr Gly Ala Ser
Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60Gly Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu65 70 75 80Pro Glu
Asp Phe Ala Val Tyr Tyr Cys Ala Thr Ile Val Ala Gly Asp 85 90 95His
Tyr Phe Asp His Trp Gly Gln Gly Thr Lys Val Glu Ile Lys Ala 100 105
110Asp Gly Ser Asp Tyr Lys Asp Asp Asp Asp Lys Ala Ser Ala 115 120
125283126PRTArtificial Sequencebinding polypeptide 283Glu Ile Val
Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15Glu
Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn 20 25
30Ser Val Ala Trp Tyr Arg Lys Lys Pro Gly Gln Ala Pro Arg Leu Leu
35 40 45Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe
Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg
Leu Glu65 70 75 80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Ala Thr Ile
Val Ala Gly Asp 85 90 95His Tyr Phe Asp His Trp Gly Gln Gly Thr Lys
Val Glu Ile Lys Ala 100 105 110Asp Gly Ser Asp Tyr Lys Asp Asp Asp
Asp Lys Ala Ser Ala 115 120 125284126PRTArtificial Sequencebinding
polypeptide 284Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu
Ser Pro Gly 1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln
Ser Val Ser Ser Asn 20 25 30Ser Val Ala Trp Tyr Arg Lys Lys Pro Gly
Gln Ala Pro Arg Leu Leu 35 40 45Ile Tyr Gly Ala Ser Ser Arg Ala Thr
Gly Ile Pro Asp Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Arg Leu Glu65 70 75 80Pro Glu Asp Phe Ala Val
Tyr Tyr Cys Ala Thr Ile Val Ala Gly Asp 85 90 95His Tyr Phe Asp His
Trp Gly Gln Gly Thr Lys Val Glu Ile Lys Ala 100 105 110Asp Gly Ser
Asp Tyr Lys Asp Asp Asp Asp Lys Ala Ser Ala 115 120
125285126PRTArtificial Sequencebinding polypeptide 285Glu Ile Val
Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15Glu
Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn 20 25
30Ser Val Ala Trp Tyr Arg Lys Lys Pro Gly Gln Ala Pro Arg Leu Leu
35 40 45Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe
Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg
Leu Glu65 70 75 80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Ala Thr Ile
Val Ala Gly Asp 85 90 95His Tyr Phe Asp His Trp Gly Gln Gly Thr Lys
Val Glu Ile Lys Ala 100 105 110Asp Gly Ser Asp Tyr Lys Asp Asp Asp
Asp Lys Ala Ser Ala 115 120 125286125PRTArtificial Sequencebinding
polypeptide 286Glu Ile Val Met Thr Gln Ser Pro Gly Thr Leu Ser Leu
Ser Pro Gly 1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln
Ser Val Ser Ser Ser 20 25 30Ser Leu Ala Trp Tyr Arg Gln Lys Pro Gly
Gln Ala Pro Arg Leu Leu 35 40 45Ile Tyr Gly Ala Ser Asn Arg Ala Thr
Gly Ile Pro Asp Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Ser Leu Gln65 70 75 80Pro Glu Asp Phe Ala Val
Tyr Tyr Cys Ala Lys Leu Thr Tyr Tyr Gly 85 90 95Ser Gly Arg Asn Trp
Gly Gln Gly Thr Lys Leu Glu Ile Lys Ala Asp 100 105 110Gly Ser Asp
Tyr Lys Asp Asp Asp Asp Lys Ala Ser Ala 115 120
125287125PRTArtificial Sequencebinding polypeptide 287Glu Ile Val
Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15Glu
Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25
30Ser Leu Ala Trp Tyr Arg Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu
35 40 45Ile Tyr Gly Ala Ser Asn Arg Ala Thr Gly Ile Pro Asp Arg Phe
Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln65 70 75 80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Ala Lys Leu
Thr Tyr Tyr Gly 85 90 95Ser Gly Arg Asn Trp Gly Gln Gly Thr Lys Leu
Glu Ile Lys Ala Asp 100 105 110Gly Ser Asp Tyr Lys Asp Asp Asp Asp
Lys Ala Ser Ala 115 120 125288125PRTArtificial Sequencebinding
polypeptide 288Glu Ile Val Met Thr Gln Ser Pro Gly Thr Leu Ser Leu
Ser Pro Gly 1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln
Ser Val Ser Ser Ser 20 25 30Ser Leu Ala Trp Tyr Arg Gln Lys Pro Gly
Gln Ala Pro Arg Leu Leu 35 40 45Ile Tyr Gly Ala Ser Asn Arg Ala Thr
Gly Ile Pro Asp Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Ser Leu Gln65 70 75 80Pro Glu Asp Phe Ala Val
Tyr Tyr Cys Ala Lys Leu Thr Tyr Tyr Gly 85 90 95Ser Gly Arg Asn Trp
Gly Gln Gly Thr Lys Leu Glu Ile Lys Ala Asp 100 105 110Gly Ser Asp
Tyr Lys Asp Asp Asp Asp Lys Ala Ser Ala 115 120
125289125PRTArtificial Sequencebinding polypeptide 289Glu Ile Val
Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15Glu
Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25
30Ser Leu Ala Trp Tyr Arg Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu
35 40 45Ile Tyr Gly Ala Ser Asn Arg Ala Thr Gly Ile Pro Asp Arg Phe
Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln65 70 75 80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Ala Lys Leu
Thr Tyr Tyr Gly 85 90 95Ser Gly Arg Asn Trp Gly Gln Gly Thr Lys Leu
Glu Ile Lys Glu Asp 100 105 110Gly Ser Asp Tyr Lys Asp Asp Asp Asp
Lys Ala Ser Ala 115 120 125290125PRTArtificial Sequencebinding
polypeptide 290Glu Ile Val Met Thr Gln Ser Pro Gly Thr Leu Ser Leu
Ser Pro Gly 1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln
Ser Val Ser Ser Ser 20 25 30Ser Leu Ala Trp Tyr Arg Gln Lys Pro Gly
Gln Ala Pro Arg Leu Leu 35 40 45Ile Tyr Gly Ala Ser Asn Arg Ala Thr
Gly Ile Pro Asp Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Ser Leu Gln65 70 75 80Pro Glu Asp Phe Ala Val
Tyr Tyr Cys Ala Lys Leu Thr Tyr Tyr Gly 85 90 95Ser Gly Arg Asn Trp
Gly Gln Gly Thr Lys Leu Glu Ile Lys Ala Asp 100 105 110Gly Ser Asp
Tyr Lys Asp Asp Asp Asp Lys Ala Ser Ala 115 120
125291125PRTArtificial Sequencebinding polypeptide 291Glu Ile Val
Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15Glu
Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25
30Ser Leu Ala Trp Tyr Arg Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu
35 40 45Ile Tyr Gly Ala Ser Asn Arg Ala Thr Gly Ile Pro Asp Arg Phe
Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln65 70 75 80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Ala Lys Leu
Thr Tyr Tyr Gly 85 90 95Ser Gly Arg Asn Trp Gly Gln Gly Thr Lys Leu
Glu Ile Lys Ala Asp 100 105 110Gly Ser Asp Tyr Lys Asp Asp Asp Asp
Lys Ala Ser Ala 115 120 125292125PRTArtificial Sequencebinding
polypeptide 292Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu
Ser Pro Gly 1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln
Ser Val Ser Ser Ser 20 25 30Ser Leu Ala Trp Tyr Arg Gln Lys Pro Gly
Gln Ala Pro Arg Leu Leu 35 40 45Ile Tyr Gly Ala Ser Asn Arg Ala Thr
Gly Ile Pro Asp Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Ser Leu Gln65 70 75 80Pro Glu Asp Phe Ala Val
Tyr Tyr Cys Ala Lys Leu Thr Xaa Tyr Gly 85 90 95Ser Gly Arg Asn Trp
Gly Gln Gly Thr Lys Leu Glu Ile Lys Ala Asp 100 105 110Gly Ser Asp
Tyr Lys Asp Asp Asp Asp Lys Ala Ser Ala 115 120
125293125PRTArtificial Sequencebinding polypeptide 293Glu Ile Val
Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15Glu
Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25
30Ser Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu
35 40 45Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe
Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg
Leu Glu65 70 75 80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Ala Lys Leu
Thr Tyr Tyr Gly 85 90 95Ser Gly Arg Asn Trp Gly Gln Gly Thr Lys Leu
Glu Ile Lys Ala Asp 100 105 110Gly Ser Asp Tyr Lys Asp Asp Asp Asp
Lys Ala Ser Ala 115 120 125294126PRTArtificial Sequencebinding
polypeptide 294Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val
Ser Pro Gly 1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln
Ser Val Gly Ile Lys 20 25 30Leu Ala Trp Tyr Gln Gln Lys Leu Gly Gln
Ala Pro Arg Leu Leu Ile 35 40 45Tyr Asp Ala Ser Thr Arg Ala Thr Gly
Val Pro Ala Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Glu Phe Thr
Leu Thr Ile Ser Ser Leu Gln Ser65 70 75 80Glu Asp Phe Ala Val Tyr
Tyr Cys Thr Gly Gly Thr Gly Asn Tyr Tyr 85 90 95Gly Trp Phe Asp Pro
Trp Gly Gln Gly Thr Lys Leu Glu Ile Lys Ala 100 105 110Asp Gly Ser
Asp Tyr Lys Asp Asp Asp Asp Lys Ala Ser Ala 115 120
125295126PRTArtificial Sequencebinding polypeptide 295Ala Ile Gln
Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15Glu
Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Arg Thr Lys 20 25
30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Arg Leu Leu Ile
35 40 45Tyr Asp Ala Ser Thr Arg Ala Thr Gly Val Pro Ala Arg Phe Ser
Gly 50 55 60Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu
Gln Pro65 70 75 80Glu Asp Phe Ala Val Tyr Tyr Cys Thr Gly Gly Thr
Gly Asn Tyr Tyr 85 90 95Gly Trp Phe Asp Pro Trp Gly Gln Gly Thr Lys
Val Glu Ile Lys Ala 100 105 110Asp Gly Ser Asp Tyr Lys Asp Asp Asp
Asp Lys Ala Ser Ala 115 120 125296125PRTArtificial Sequencebinding
polypeptide 296Ala Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala
Ser Val Gly 1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
Ser Ile Ser Thr Tyr 20 25 30Leu Asn Trp Tyr Gln Leu Lys Pro Gly Glu
Ala Pro Lys Arg Leu Ile 35 40 45Tyr Ala Ala Ser Asn Leu Glu Ser Gly
Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Glu Phe Thr
Leu Thr Ile Asn Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Val Tyr
Tyr Cys Ala Gly Ser Phe Ile Ala Arg Gly 85 90 95Pro Leu Asn Tyr Trp
Gly Gln Gly Thr Lys Val Glu Ile Lys Ala Asp 100 105 110Gly Ser Asp
Tyr Lys Asp Asp Asp Asp Lys Ala Ser Ala 115 120
125297125PRTArtificial Sequencebinding polypeptide 297Ala Ile Gln
Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Thr Tyr 20 25
30Leu Asn Trp Tyr Gln Leu Lys Pro Gly Glu Ala Pro Lys Arg Leu Ile
35 40 45Tyr Ala Ala Ser Asn Leu Glu Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Asn Ser Leu
Gln Pro65 70 75 80Glu Asp Phe Ala Val Tyr Tyr Cys Ala Gly Ser Phe
Ile Ala Arg Gly 85 90 95Pro Leu Asn Tyr Trp Gly Gln Gly Thr Lys Val
Glu Ile Lys Ala Asp 100 105 110Gly Ser Asp Tyr Lys Asp Asp Asp Asp
Lys Ala Ser Ala 115 120 125298128PRTArtificial Sequencebinding
polypeptide 298Ser Tyr Glu Leu Thr Gln Pro Ala Ser Val Ser Gly Ser
Pro Gly Gln 1 5 10 15Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser
Asp Val Gly Gly Tyr 20 25 30Asp His Val Phe Trp Tyr Gln Gln His Pro
Gly Lys Ala Pro Lys Leu 35 40 45Met Ile Tyr Glu Asp Ser Lys Arg Pro
Ser Gly Val Ser Asn Arg Phe 50 55 60Ser Gly Ser Lys Ser Gly Asn Thr
Ala Ser Leu Thr Ile Ser Gly Leu65 70 75 80Gln Asp Glu Asp Glu Ala
Val Tyr Tyr Cys Ala Lys Arg Arg Tyr Val 85 90 95Asn Ala Gly Trp Phe
Asp Tyr Trp Gly Gln Gly Thr Lys Leu Thr Val 100 105 110Leu Ala Asp
Gly Ser Asp Tyr Lys Asp Asp Asp Asp Lys Ala Ser Ala 115 120
125299128PRTArtificial Sequencebinding polypeptide 299Gln Ser Ala
Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15Ser
Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr 20 25
30Asp His Val Phe Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu
35 40 45Met Ile Tyr Glu Asp Ser Lys Arg Pro Ser Gly Val Ser Asn Arg
Phe 50 55 60Ser Gly Ser Lys Ser
Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu65 70 75 80Gln Ala Glu
Asp Glu Ala Val Tyr Tyr Cys Ala Lys Arg Arg Tyr Val 85 90 95Asn Ala
Gly Trp Phe Asp Tyr Trp Gly Gln Gly Thr Lys Leu Thr Val 100 105
110Leu Ala Asp Gly Ser Asp Tyr Lys Asp Asp Asp Asp Lys Ala Ser Ala
115 120 125300128PRTArtificial Sequencebinding polypeptide 300Gln
Ser Val Leu Thr Gln Pro Arg Ser Val Ser Gly Ser Pro Gly Gln 1 5 10
15Ser Val Ala Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr
20 25 30Asn Tyr Val Phe Trp Tyr Gln Arg His Pro Gly Lys Ala Pro Lys
Leu 35 40 45Ile Ile Tyr Glu Asp Asn Lys Arg Pro Ser Gly Val Pro Asp
Arg Phe 50 55 60Ser Gly Ser Lys Phe Gly Asn Thr Ala Ser Leu Thr Ile
Ser Gly Leu65 70 75 80His Thr Glu Asp Glu Ala Val Tyr Tyr Cys Ala
Lys Arg Arg Tyr Val 85 90 95Asn Ala Gly Trp Phe Asp Tyr Trp Gly Gln
Gly Thr Lys Val Thr Val 100 105 110Leu Ala Asp Gly Ser Asp Tyr Lys
Asp Asp Asp Asp Lys Ala Ser Ala 115 120 125301128PRTArtificial
Sequencebinding polypeptide 301Gln Ser Val Leu Thr Gln Pro Arg Ser
Val Ser Gly Ser Pro Gly Gln 1 5 10 15Ser Val Ala Ile Ser Cys Thr
Gly Thr Ser Ser Asp Val Gly Gly Tyr 20 25 30Asn Tyr Val Phe Trp Tyr
Gln Arg His Pro Gly Lys Ala Pro Lys Leu 35 40 45Ile Ile Tyr Glu Asp
Asn Lys Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60Ser Gly Ser Lys
Phe Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu65 70 75 80His Thr
Glu Asp Glu Ala Val Tyr Tyr Cys Ala Lys Arg Arg Tyr Val 85 90 95Asn
Ala Gly Trp Phe Asp Tyr Trp Gly Gln Gly Thr Lys Val Thr Val 100 105
110Leu Ala Asp Gly Ser Asp Tyr Lys Asp Asp Asp Asp Lys Ala Ser Ala
115 120 125302128PRTArtificial Sequencebinding polypeptide 302Gln
Ser Val Leu Thr Gln Pro Arg Ser Val Ser Gly Ser Pro Gly Gln 1 5 10
15Ser Val Ala Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr
20 25 30Asn Tyr Val Phe Trp Tyr Gln Arg His Pro Gly Lys Ala Pro Lys
Leu 35 40 45Ile Ile Tyr Glu Asp Asn Lys Arg Pro Ser Gly Val Pro Asp
Arg Phe 50 55 60Ser Gly Ser Lys Phe Gly Asn Thr Ala Ser Leu Thr Ile
Ser Gly Leu65 70 75 80His Thr Glu Asp Glu Ala Val Tyr Tyr Cys Ala
Lys Arg Arg Tyr Val 85 90 95Asn Ala Gly Trp Phe Asp Tyr Trp Gly Gln
Gly Thr Lys Val Thr Val 100 105 110Leu Ala Asp Gly Ser Asp Tyr Lys
Asp Asp Asp Asp Lys Ala Ser Ala 115 120 125303128PRTArtificial
Sequencebinding polypeptide 303Gln Ser Val Leu Thr Gln Pro Arg Ser
Val Ser Gly Ser Pro Gly Gln 1 5 10 15Ser Val Ala Ile Ser Cys Thr
Gly Thr Ser Ser Asp Val Gly Gly Tyr 20 25 30Asn Tyr Val Phe Trp Tyr
Gln Arg His Pro Gly Lys Ala Pro Lys Leu 35 40 45Ile Ile Tyr Glu Asp
Asn Lys Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60Ser Gly Ser Lys
Phe Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu65 70 75 80His Thr
Glu Asp Glu Ala Val Tyr Tyr Cys Ala Lys Arg Arg Tyr Val 85 90 95Asn
Ala Gly Trp Phe Asp Tyr Trp Gly Gln Gly Thr Lys Val Thr Val 100 105
110Leu Ala Asp Gly Ser Asp Tyr Lys Asp Asp Asp Asp Lys Ala Ser Ala
115 120 125304128PRTArtificial Sequencebinding polypeptide 304Ser
Tyr Glu Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln 1 5 10
15Ser Val Ala Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr
20 25 30Asn Tyr Val Phe Trp Tyr Gln Arg His Pro Gly Lys Ala Pro Lys
Leu 35 40 45Ile Ile Tyr Glu Asp Asn Lys Arg Pro Ser Gly Val Pro Asp
Arg Phe 50 55 60Ser Gly Ser Lys Phe Gly Asn Thr Ala Ser Leu Thr Ile
Ser Gly Leu65 70 75 80His Thr Glu Asp Glu Ala Val Tyr Tyr Cys Ala
Lys Arg Arg Tyr Val 85 90 95Asn Ala Gly Trp Phe Asp Tyr Trp Gly Gln
Gly Thr Lys Val Thr Val 100 105 110Leu Ala Asp Gly Ser Asp Tyr Lys
Asp Asp Asp Asp Lys Ala Ser Ala 115 120 125305127PRTArtificial
Sequencebinding polypeptide 305Gln Ser Val Leu Thr Gln Pro Pro Ser
Val Ser Gly Ala Pro Gly Gln 1 5 10 15Arg Val Thr Ile Ser Cys Thr
Gly Ser Ser Ser Asn Ile Gly Ala Gly 20 25 30Tyr Asp Val His Trp Tyr
Gln Gln Leu Pro Gly Ala Ala Pro Lys Leu 35 40 45Leu Ile Tyr Gly Asn
Ser Asn Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60Ser Gly Ser Lys
Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu65 70 75 80Gln Ala
Glu Asp Glu Ala Val Tyr Tyr Cys Ala Ser Ala Ile Pro Gly 85 90 95Thr
Arg Ile Leu Arg Asp Trp Gly Gln Gly Thr Lys Leu Thr Val Leu 100 105
110Ala Asp Gly Ser Asp Tyr Lys Asp Asp Asp Asp Lys Ala Ser Ala 115
120 125306123PRTArtificial Sequencebinding polypeptide 306Glu Ile
Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10
15Glu Arg Ala Ala Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Asp Lys
20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu
Ile 35 40 45Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe
Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Glu Pro65 70 75 80Glu Asp Phe Ala Val Tyr Tyr Cys Ala Thr Glu
Phe Arg Gly Tyr Ala 85 90 95Gly Tyr Trp Gly Gln Gly Thr Lys Leu Glu
Ile Lys Ala Asp Gly Ser 100 105 110Asp Tyr Lys Asp Asp Asp Asp Lys
Ala Ser Ala 115 120307123PRTArtificial Sequencebinding polypeptide
307Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly
1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser
Ser Asn 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg
Leu Leu Ile 35 40 45Tyr Gly Ala Ser Arg Arg Ala Thr Gly Ile Pro Asp
Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
Ser Arg Leu Glu Pro65 70 75 80Glu Asp Phe Ala Val Tyr Tyr Cys Ala
Gly Phe Asp Pro Tyr Gln Pro 85 90 95Pro Arg Trp Gly Gln Gly Thr Lys
Leu Glu Ile Lys Ala Asp Gly Ser 100 105 110Asp Tyr Lys Asp Asp Asp
Asp Lys Ala Ser Ala 115 120308123PRTArtificial Sequencebinding
polypeptide 308Glu Ile Val Leu Thr Gln Ser Pro Asp Phe Gln Ser Val
Thr Pro Lys 1 5 10 15Glu Lys Val Thr Ile Thr Cys Arg Ala Ser Gln
Ser Ile Val Thr Ser 20 25 30Leu His Trp Tyr Gln Gln Lys Pro Gly Gln
Ala Pro Ser Leu Leu Ile 35 40 45Tyr Gly Ala Ser Asn Arg Ala Thr Gly
Ile Pro Ala Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Glu Phe Thr
Leu Thr Ile Ser Gly Leu Gln Pro65 70 75 80Glu Asp Ala Ala Val Tyr
Tyr Cys Ala Gly Asp Phe Gly Gly Gln Leu 85 90 95Pro Tyr Trp Gly Gln
Gly Thr Lys Leu Glu Ile Lys Ala Asp Gly Ser 100 105 110Asp Tyr Lys
Asp Asp Asp Asp Lys Ala Ser Ala 115 120309129PRTArtificial
Sequencebinding polypeptide 309Asp Ile Val Met Thr Gln Ser Pro Leu
Ser Leu Pro Val Thr Leu Gly 1 5 10 15Gln Pro Ala Ser Ile Ser Cys
Arg Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30Asp Gly Asn Thr Tyr Leu
Asn Trp Phe Gln Gln Arg Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu Ile
Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro 50 55 60Asp Arg Phe Ser
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg
Val Glu Ala Glu Asp Val Ala Val Tyr Tyr Cys Ala Arg Ala 85 90 95Gly
Ser Ser Trp Lys Phe Asp Tyr Trp Gly Gln Gly Thr Lys Val Glu 100 105
110Ile Lys Ala Asp Gly Ser Asp Tyr Lys Asp Asp Asp Asp Lys Ala Ser
115 120 125Ala310129PRTArtificial Sequencebinding polypeptide
310Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly
1 5 10 15Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu
Leu Thr 20 25 30Ser Gly Asp Asn Tyr Leu Asn Trp Tyr Leu Gln Lys Pro
Gly Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala
Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly
Val Tyr Tyr Cys Ala Arg Met 85 90 95Gly Ala Val Ala Gly Tyr Asp Tyr
Trp Gly Gln Gly Thr Lys Val Glu 100 105 110Ile Lys Ala Asp Gly Ser
Asp Tyr Lys Asp Asp Asp Asp Lys Ala Ser 115 120
125Ala31186PRTArtificial Sequencebinding polypeptide 311Glu Val Val
Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro
His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25
30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr
35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr
Gly 50 55 60Tyr Ala Val Thr Val Glu Gln Asp Gly His Val Leu Tyr Ile
Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 8531286PRTArtificial
Sequencebinding polypeptide 312Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Met Gly Lys Asn Gly His Glu Leu Leu Thr Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 8531386PRTArtificial Sequencebinding polypeptide
313Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His
1 5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln
Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr
Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr Pro Gly Pro Gly Asp Arg Glu
Leu Ile Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
8531486PRTArtificial Sequencebinding polypeptide 314Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Ala Gly Pro Gly Ala His Glu Leu Leu Thr Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 8531586PRTArtificial
Sequencebinding polypeptide 315Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Met Ala Gln Asn Asn Arg Glu Leu Leu Thr Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 8531686PRTArtificial Sequencebinding polypeptide
316Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His
1 5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln
Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr
Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr Met Ala Gln Tyr Gly Arg Glu
Leu Leu Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
8531786PRTArtificial Sequencebinding polypeptide 317Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Leu Ala His Asn Gly Asn Glu Leu Leu Thr Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 8531886PRTArtificial
Sequencebinding polypeptide 318Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Val Ala Trp Asn Gly His Glu Leu Met Thr Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 8531986PRTArtificial Sequencebinding polypeptide
319Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His
1 5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln
Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr
Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr Leu Gly Leu Arg Asp Arg Glu
Leu Phe Val Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
8532086PRTArtificial Sequencebinding polypeptide 320Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Ser Gly Leu Asn Asp Arg Val Leu Phe Ile Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 8532186PRTArtificial
Sequencebinding polypeptide 321Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr
Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val
Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly
Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr Met Gly Pro Asn
Asp Arg Glu Leu Leu Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
8532286PRTArtificial Sequencebinding polypeptide 322Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Leu Gly His Asn Asp Arg Glu Leu Leu Thr Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 8532386PRTArtificial
Sequencebinding polypeptide 323Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Leu Gly Leu Asn Asp Arg Glu Leu Met Thr Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 8532486PRTArtificial Sequencebinding polypeptide
324Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His
1 5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln
Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr
Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr Met Ala Gln Asn Gly His Lys
Leu Met Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
8532586PRTArtificial Sequencebinding polypeptide 325Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Val His Trp Asn Gly His Glu Leu Met Thr Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 8532686PRTArtificial
Sequencebinding polypeptide 326Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Met Gly Phe Met Ala His Glu Leu Met Val Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 8532786PRTArtificial Sequencebinding polypeptide
327Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His
1 5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln
Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr
Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr Ala Gly Leu Asn Glu His Glu
Leu Leu Ile Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
8532886PRTArtificial Sequencebinding polypeptide 328Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Leu Ala Asp Asn Ala Arg Glu Leu Leu Thr Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 8532986PRTArtificial
Sequencebinding polypeptide 329Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Leu Gly Lys Asp Val Arg Glu Leu Leu Thr Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 8533086PRTArtificial Sequencebinding polypeptide
330Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His
1 5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln
Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr
Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr Leu Ser Asp Ser Gly His Ala
Leu Phe Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
8533186PRTArtificial Sequencebinding polypeptide 331Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Leu Gly Pro Tyr Glu His Glu Leu Leu Thr Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 8533286PRTArtificial
Sequencebinding polypeptide 332Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Ala Gly Arg His Asp His Glu Leu Ile Ile Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 8533386PRTArtificial Sequencebinding polypeptide
333Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His
1 5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln
Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr
Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr Ile Gly Pro Asn Asn His Glu
Leu Leu Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
8533486PRTArtificial Sequencebinding polypeptide 334Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Val Glu Gln Asn Gly Arg Glu Leu Ile Ile Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 8533586PRTArtificial
Sequencebinding polypeptide 335Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Ala Gly Leu Asp Glu His Glu Leu Leu Ile Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 8533686PRTArtificial Sequencebinding polypeptide
336Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His
1 5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln
Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr
Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr Val Ala Pro Asn Gly His Glu
Leu Phe Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
8533786PRTArtificial Sequencebinding polypeptide 337Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Met Ala Gln Asn Gly His Ala Leu Phe Thr Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 8533886PRTArtificial
Sequencebinding polypeptide 338Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Val Gly Tyr Asn Asn Arg Glu Leu Leu Thr Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 8533986PRTArtificial Sequencebinding polypeptide
339Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His
1 5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln
Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr
Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr Val Ala Gln Asp Gly His Phe
Leu Tyr Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
8534086PRTArtificial Sequencebinding polypeptide 340Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Ser Gly His Asn Gly His Glu Val Met Thr Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 8534186PRTArtificial
Sequencebinding polypeptide 341Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Phe Asp Gln Ser Asp His Glu Leu Leu Thr Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 8534286PRTArtificial Sequencebinding polypeptide
342Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His
1 5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln
Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr
Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr Val Gly Pro Asn Glu Arg Met
Leu Met Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
8534386PRTArtificial Sequencebinding polypeptide 343Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Gly Tyr Tyr Asn Asp Arg Glu Leu Leu Thr Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 8534486PRTArtificial
Sequencebinding polypeptide 344Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Leu Thr His Asn Asp His Glu Leu Leu Thr Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 8534586PRTArtificial Sequencebinding polypeptide
345Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His
1 5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln
Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr
Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr Val Gly Arg Asn Asp Arg Glu
Leu Leu Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
8534686PRTArtificial Sequencebinding polypeptide 346Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Trp Ala Gln Asn Gly Arg Glu Leu Leu Thr Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 8534786PRTArtificial
Sequencebinding polypeptide 347Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Leu Gly Lys Asn Asp His Glu Leu Leu Thr Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 8534886PRTArtificial Sequencebinding polypeptide
348Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His
1 5
10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro
Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr
Ile Thr Gly 50 55 60Tyr Ala Val Thr Leu Gly Pro Asn Asp His Glu Leu
Met Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
8534986PRTArtificial Sequencebinding polypeptide 349Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Thr Gly Trp Asn Gly Asn Glu Leu Phe Thr Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 8535086PRTArtificial
Sequencebinding polypeptide 350Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Leu Ala His Asn Asp His Glu Leu Leu Thr Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 8535186PRTArtificial Sequencebinding polypeptide
351Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His
1 5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln
Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr
Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr Leu Glu Gln Asn Asp Arg Val
Leu Leu Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
8535286PRTArtificial Sequencebinding polypeptide 352Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Thr Gly His His Asp His Glu Leu Ile Ile Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 8535386PRTArtificial
Sequencebinding polypeptide 353Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Val Ala His Glu Asn Arg Glu Leu Leu Thr Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 8535486PRTArtificial Sequencebinding polypeptide
354Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His
1 5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln
Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr
Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr Leu Gly Leu Asn Asp His Glu
Leu Ile Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
8535586PRTArtificial Sequencebinding polypeptide 355Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Ala Gly Pro Asn Asp His Gln Leu Phe Thr Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 8535686PRTArtificial
Sequencebinding polypeptide 356Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Asp Ala Met Tyr Gly Arg Glu Leu Met Thr Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 8535786PRTArtificial Sequencebinding polypeptide
357Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His
1 5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln
Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr
Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr Val Ala Trp Asp Asp His Glu
Leu Leu Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
8535886PRTArtificial Sequencebinding polypeptide 358Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Met Gly Gln Asn Asp Lys Glu Leu Ile Thr Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 8535986PRTArtificial
Sequencebinding polypeptide 359Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Leu Ala Gln Asn Gly His Glu Leu Tyr Thr Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 8536086PRTArtificial Sequencebinding polypeptide
360Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His
1 5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln
Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr
Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr Pro Gly His Asn Asp His Glu
Leu Met Val Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
8536186PRTArtificial Sequencebinding polypeptide 361Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Glu Ala Arg Asn Gly Arg Glu Leu Leu Thr Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 8536286PRTArtificial
Sequencebinding polypeptide 362Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Leu Ala His Asn Asp Arg Glu Leu Leu Thr Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 8536386PRTArtificial Sequencebinding polypeptide
363Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His
1 5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln
Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr
Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr Met Ala His Asn Asp His Glu
Leu Leu Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
8536486PRTArtificial Sequencebinding polypeptide 364Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Leu Gly Gln Asn Asp Arg Gln Leu Leu Val Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 8536586PRTArtificial
Sequencebinding polypeptide 365Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Ala Gly Gly Asn Gly His Glu Leu Leu Thr Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 8536686PRTArtificial Sequencebinding polypeptide
366Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His
1 5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln
Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr
Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr His Gly Pro Tyr Asp Gln Val
Leu Leu Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
8536786PRTArtificial Sequencebinding polypeptide 367Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Ile Glu Gln Ser Gly Leu Gln Leu Met Thr Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 8536886PRTArtificial
Sequencebinding polypeptide 368Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Leu Ala Gln Asn Asp Arg Glu Leu Leu Thr Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 8536986PRTArtificial Sequencebinding polypeptide
369Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His
1 5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln
Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr
Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr Val Ala Trp Asp Gly Arg Glu
Leu Phe Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
8537086PRTArtificial Sequencebinding polypeptide 370Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Leu Ala Tyr Asn Gly Arg Glu Ile Ile Thr Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 8537186PRTArtificial
Sequencebinding polypeptide 371Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Trp Ser Gln Asn Asn Arg Glu Leu Phe Thr Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 8537286PRTArtificial Sequencebinding polypeptide
372Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His
1 5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln
Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr
Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr Glu Thr Trp Asn Asp His Glu
Ile Arg Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
8537386PRTArtificial Sequencebinding polypeptide 373Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Val Ala Gln Asn Gly His Gln Leu Phe Thr Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 8537494PRTArtificial
Sequencebinding polypeptide 374Val Ser Asp Val Pro Arg Asp Leu Glu
Val Val Ala Ala Thr Pro Thr 1 5 10 15Ser Leu Leu Ile Ser Trp Arg
His Pro His Phe Pro Thr Arg Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 35 40 45Thr Val Pro Leu Gln
Pro Pro Thr Ala Thr Ile Ser Gly Leu Lys Pro 50 55 60Gly Val Asp Tyr
Thr Ile Thr Gly Tyr Ala Val Thr Val Thr His Asn65 70 75 80Gly His
Pro Leu Met Thr Pro Ile Ser Ile Asn Tyr Arg Thr 85
90375107PRTArtificial
Sequencebinding polypeptide 375Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Tyr Tyr Arg Ile Thr 35 40 45Tyr Gly Glu Thr Gly
Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro 50 55 60Leu Gln Pro Pro
Thr Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp65 70 75 80Tyr Thr
Ile Thr Gly Tyr Ala Val Thr Phe Ala Gln Asn Asp His Gln 85 90 95Leu
Phe Thr Pro Ile Ser Ile Asn Tyr Arg Thr 100 105376107PRTArtificial
Sequencebinding polypeptide 376Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Tyr Tyr Arg Ile Thr 35 40 45Tyr Gly Glu Thr Gly
Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro 50 55 60Leu Gln Pro Pro
Thr Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp65 70 75 80Tyr Thr
Ile Thr Gly Tyr Ala Val Thr Gly Gly Gln Met Asn Arg Val 85 90 95Leu
Met Thr Pro Ile Ser Ile Asn Tyr Arg Thr 100 105377107PRTArtificial
Sequencebinding polypeptide 377Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Tyr Tyr Arg Ile Thr 35 40 45Tyr Gly Glu Thr Gly
Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro 50 55 60Leu Gln Pro Pro
Thr Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp65 70 75 80Tyr Thr
Ile Thr Gly Tyr Ala Val Thr Leu Val His Asn Asp Arg Glu 85 90 95Leu
Leu Thr Pro Ile Ser Ile Asn Tyr Arg Thr 100 105378107PRTArtificial
Sequencebinding polypeptide 378Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Tyr Tyr Arg Ile Thr 35 40 45Tyr Gly Glu Thr Gly
Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro 50 55 60Leu Gln Pro Pro
Thr Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp65 70 75 80Tyr Thr
Ile Thr Gly Tyr Ala Val Thr Val Ala Gln Asn Gly His Glu 85 90 95Leu
Phe Thr Pro Ile Ser Ile Asn Tyr Arg Thr 100 105379115PRTArtificial
Sequencebinding polypeptide 379Val Ser Asp Val Pro Arg Asp Leu Glu
Val Val Ala Ala Thr Pro Thr 1 5 10 15Ser Leu Leu Ile Ser Trp Arg
His Pro His Phe Pro Thr Arg Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 35 40 45Thr Val Pro Tyr Tyr
Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser 50 55 60Pro Val Gln Glu
Phe Thr Val Pro Leu Gln Pro Pro Thr Ala Thr Ile65 70 75 80Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly Tyr Ala Val 85 90 95Thr
Val His Trp Asn Gly His Glu Leu Met Thr Pro Ile Ser Ile Asn 100 105
110Tyr Arg Thr 115380107PRTArtificial Sequencebinding polypeptide
380Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His
1 5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Tyr Tyr
Arg Ile Thr 35 40 45Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu
Phe Thr Val Pro 50 55 60Leu Gln Pro Pro Thr Ala Thr Ile Ser Gly Leu
Lys Pro Gly Val Asp65 70 75 80Tyr Thr Ile Thr Gly Tyr Ala Val Thr
Leu Gly Trp Asn Asp His Glu 85 90 95Leu Tyr Ile Pro Ile Ser Ile Asn
Tyr Arg Thr 100 10538186PRTArtificial Sequencebinding polypeptide
381Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His
1 5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln
Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr
Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr Ala Gly His Lys Asp Gln Glu
Leu Leu Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
8538286PRTArtificial Sequencebinding polypeptide 382Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Leu Ala Gln Asn Asn His Glu Leu Leu Thr Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 8538386PRTArtificial
Sequencebinding polypeptide 383Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Val Ala Trp Asn Asp His Glu Ile Tyr Thr Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 8538486PRTArtificial Sequencebinding polypeptide
384Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His
1 5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln
Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr
Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr Leu Ala Gln Thr Gly Arg Glu
Leu Leu Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
8538586PRTArtificial Sequencebinding polypeptide 385Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Val Gly Trp Ser Gly His Glu Leu Phe Val Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 8538686PRTArtificial
Sequencebinding polypeptide 386Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Val Gly His Asn Asp Arg Glu Leu Ile Thr Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 8538786PRTArtificial Sequencebinding polypeptide
387Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His
1 5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln
Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr
Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr Trp Asn Gln Asn Gly Gln Glu
Leu Phe Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
8538886PRTArtificial Sequencebinding polypeptide 388Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Phe Gly Gln Asn Gly His Ala Leu Leu Thr Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 8538986PRTArtificial
Sequencebinding polypeptide 389Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Arg Gly Leu Asn Asp Gly Glu Leu Leu Thr Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 8539086PRTArtificial Sequencebinding polypeptide
390Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His
1 5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln
Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr
Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr Phe Gly Pro Ser Asp His Val
Leu Leu Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
8539186PRTArtificial Sequencebinding polypeptide 391Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Leu Ala Gln Asn Asn His Glu Leu Leu Thr Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 8539286PRTArtificial
Sequencebinding polypeptide 392Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Val Ala Gln Asn Asp His Lys Leu Phe Ile Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 8539386PRTArtificial Sequencebinding polypeptide
393Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His
1 5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln
Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr
Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr Arg Asp Gln Tyr Glu His Glu
Leu Leu Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
8539486PRTArtificial Sequencebinding polypeptide 394Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Leu Ala Leu Asn Gly His Glu Leu Phe Thr Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 8539586PRTArtificial
Sequencebinding polypeptide 395Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Val Glu Ser Asn Gly His Ala Leu Phe Val Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 8539686PRTArtificial Sequencebinding polypeptide
396Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His
1 5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln
Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr
Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr Val Gly Gln Asn Asn His Glu
Leu Leu Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
8539786PRTArtificial Sequencebinding polypeptide 397Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Trp Asp Gln Asn Gly His Val Leu Leu Thr Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 8539886PRTArtificial
Sequencebinding polypeptide 398Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Glu Gly Leu Asn Asp His Glu Leu Ile Ile Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 8539986PRTArtificial Sequencebinding polypeptide
399Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His
1 5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln
Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr
Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr Glu Gly Leu Asn Asp His Glu
Leu Met Ile Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
8540086PRTArtificial Sequencebinding polypeptide 400Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25
30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr
35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr
Gly 50 55 60Tyr Ala Val Thr Glu Gly Gln Asn Asp Gln Leu Leu Phe Ile
Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 8540186PRTArtificial
Sequencebinding polypeptide 401Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Leu Ala Gln Asn Gly His Glu Leu Leu Thr Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 8540286PRTArtificial Sequencebinding polypeptide
402Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His
1 5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln
Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr
Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr Val Ala Gln Asn Asp Arg Glu
Leu Leu Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
8540386PRTArtificial Sequencebinding polypeptide 403Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Leu Ala Gln Asn Gly His Glu Leu Phe Thr Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 8540486PRTArtificial
Sequencebinding polypeptide 404Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Val Ala Gln Asn Glu Arg Glu Leu Phe Thr Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 8540586PRTArtificial Sequencebinding polypeptide
405Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His
1 5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln
Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr
Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr Val Ala Trp Asn Asp His Met
Leu Met Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
8540686PRTArtificial Sequencebinding polypeptide 406Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Leu Gly Pro Asn Asp Arg Glu Leu Met Thr Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 8540794PRTArtificial
Sequencebinding polypeptide 407Val Ser Asp Val Pro Arg Asp Leu Glu
Val Val Ala Ala Thr Pro Thr 1 5 10 15Ser Leu Leu Ile Ser Trp Arg
His Pro His Phe Pro Thr Arg Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 35 40 45Thr Val Pro Leu Gln
Pro Pro Thr Ala Thr Ile Ser Gly Leu Lys Pro 50 55 60Gly Val Asp Tyr
Thr Ile Thr Gly Tyr Ala Val Thr Val Gly Pro Asn65 70 75 80Glu Arg
Met Leu Met Thr Pro Ile Ser Ile Asn Tyr Arg Thr 85
9040886PRTArtificial Sequencebinding polypeptide 408Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Val Ala His Asn Asp His Glu Leu Leu Thr Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 8540986PRTArtificial
Sequencebinding polypeptide 409Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Val Ala Lys Asn Asp His Glu Leu Leu Thr Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 8541086PRTArtificial Sequencebinding polypeptide
410Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His
1 5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln
Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr
Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr Trp Ala Gln Asn Asp His Glu
Leu Leu Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
8541186PRTArtificial Sequencebinding polypeptide 411Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Phe Ala Gln Asn Asp His Glu Leu Leu Thr Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 8541286PRTArtificial
Sequencebinding polypeptide 412Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Met Glu Gln Asn Gly His Glu Leu Phe Thr Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 8541386PRTArtificial Sequencebinding polypeptide
413Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His
1 5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln
Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr
Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr Asp Ala Pro Asn Gly Arg Glu
Leu Met Val Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
8541486PRTArtificial Sequencebinding polypeptide 414Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Gly Gly Arg Asn Gly His Thr Leu Leu Thr Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 8541586PRTArtificial
Sequencebinding polypeptide 415Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Leu Ser Gln Thr Asp His Glu Leu Leu Thr Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 8541686PRTArtificial Sequencebinding polypeptide
416Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His
1 5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln
Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr
Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr Val Gly Gln Asn Glu His Glu
Leu Leu Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
8541786PRTArtificial Sequencebinding polypeptide 417Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Val Ala Gln Asn Gly His Glu Leu Lys Thr Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 8541886PRTArtificial
Sequencebinding polypeptide 418Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Val Ala Gln Asn Asp Arg Glu Leu Phe Thr Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 8541986PRTArtificial Sequencebinding polypeptide
419Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His
1 5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln
Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr
Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr Val Gly Gln Asn His His Glu
Leu Phe Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
8542086PRTArtificial Sequencebinding polypeptide 420Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Val Gly Pro His Asp Arg Glu Leu Leu Thr Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 8542194PRTArtificial
Sequencebinding polypeptide 421Val Ser Asp Val Pro Arg Asp Leu Glu
Val Val Ala Ala Thr Pro Thr 1 5 10 15Ser Leu Leu Ile Ser Trp Arg
His Pro His Phe Pro Thr Arg Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 35 40 45Thr Val Pro Leu Gln
Pro Pro Thr Ala Thr Ile Ser Gly Leu Lys Pro 50 55 60Gly Val Asp Tyr
Thr Ile Thr Gly Tyr Ala Val Thr Met Gly Phe Met65 70 75 80Ala His
Glu Leu Met Val Pro Ile Ser Ile Asn Tyr Arg Thr 85
9042286PRTArtificial Sequencebinding polypeptide 422Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Leu Ala Gln Asn Asp His Glu Leu Leu Thr Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 8542386PRTArtificial
Sequencebinding polypeptide 423Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Leu Val Arg Asn Asp His Glu Leu Leu Thr Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 8542486PRTArtificial Sequencebinding polypeptide
424Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His
1 5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln
Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr
Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr Leu Ala Gln Asp Asp His Glu
Leu Leu Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
8542586PRTArtificial Sequencebinding polypeptide 425Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Glu Asp Ile Arg Val Leu Trp Leu Asn Thr Thr
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 8542686PRTArtificial
Sequencebinding polypeptide 426Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Val Thr Gln Asn Asp His Glu Leu Leu Thr Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 8542786PRTArtificial Sequencebinding polypeptide
427Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His
1
5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro
Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr
Ile Thr Gly 50 55 60Tyr Ala Val Thr Val Gly Gln Asn Asp His Glu Leu
Leu Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
8542886PRTArtificial Sequencebinding polypeptide 428Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Met Ala Gln Asn Asp His Lys Leu Phe Thr Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 8542986PRTArtificial
Sequencebinding polypeptide 429Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Leu Ala Gln Asn Asp His Glu Leu Leu Thr Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 8543086PRTArtificial Sequencebinding polypeptide
430Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His
1 5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln
Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr
Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr Met Ala Gln Asn Asp His Glu
Leu Leu Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
8543186PRTArtificial Sequencebinding polypeptide 431Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Val Ala Gln Asn Asn His Glu Leu Leu Thr Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 8543286PRTArtificial
Sequencebinding polypeptide 432Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Leu Ala Gln Asn Asp Arg Glu Leu Ile Thr Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 8543386PRTArtificial Sequencebinding polypeptide
433Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His
1 5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln
Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr
Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr Val Gly Gln Asn Asn His Glu
Leu Ile Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
8543486PRTArtificial Sequencebinding polypeptide 434Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Leu Ala Gln Asn Gly His Glu Leu Ile Thr Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 8543586PRTArtificial
Sequencebinding polypeptide 435Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Thr Ala His Asn Gly His Glu Leu Leu Thr Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 8543686PRTArtificial Sequencebinding polypeptide
436Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His
1 5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln
Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr
Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr Leu Gly Tyr His Asp His Ala
Leu Phe Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
8543786PRTArtificial Sequencebinding polypeptide 437Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Trp Ala Trp Asn Asp His Glu Leu Met Thr Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 8543886PRTArtificial
Sequencebinding polypeptide 438Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Val Ala Gln Asn Asp His Glu Leu Leu Thr Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 8543986PRTArtificial Sequencebinding polypeptide
439Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His
1 5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln
Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr
Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr Met Ala Gln Asn Asp His Glu
Leu Met Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
8544086PRTArtificial Sequencebinding polypeptide 440Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Met Ala Gln Asn Asp His Glu Leu Leu Thr Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 8544186PRTArtificial
Sequencebinding polypeptide 441Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Val Ala Gln Asn Gly His Glu Leu Ile Thr Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 8544294PRTArtificial Sequencebinding polypeptide
442Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr
1 5 10 15Ser Leu Leu Ile Ser Trp Arg His Pro His Phe Pro Thr Arg
Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val
Gln Glu Phe 35 40 45Thr Val Pro Leu Gln Pro Pro Thr Ala Thr Ile Ser
Gly Leu Lys Pro 50 55 60Gly Val Asp Tyr Thr Ile Thr Gly Tyr Ala Val
Thr Glu Gly Trp Ile65 70 75 80Asp His Glu Ile Met Ile Pro Ile Ser
Ile Asn Tyr Arg Thr 85 9044386PRTArtificial Sequencebinding
polypeptide 443Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser
Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr
Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val
Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly
Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr Glu Gly Gln Asn
Gly Ser Glu Leu Ile Val Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
8544486PRTArtificial Sequencebinding polypeptide 444Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Met Ala Gln Asn Asp Arg Glu Leu Ile Thr Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 8544586PRTArtificial
Sequencebinding polypeptide 445Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Val Gly Gln Asn Asp His Glu Leu Phe Thr Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 8544686PRTArtificial Sequencebinding polypeptide
446Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His
1 5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln
Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr
Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr Val Ala Gln Ser Asp His Glu
Leu Phe Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
8544786PRTArtificial Sequencebinding polypeptide 447Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Val Asp Arg Asn Asp His Glu Leu Phe Thr Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 8544886PRTArtificial
Sequencebinding polypeptide 448Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Leu Ala Gln Asn Asp His Glu Leu Met Thr Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 8544986PRTArtificial Sequencebinding polypeptide
449Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His
1 5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln
Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr
Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr Val Ala Gln Asn Asp His Glu
Leu Phe Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
8545086PRTArtificial Sequencebinding polypeptide 450Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Leu Gly Glu Asn Asp Arg Lys Leu Ile Thr Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 8545186PRTArtificial
Sequencebinding polypeptide 451Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Val Ala Gln Asn Asp His Glu Leu Leu Thr Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 8545286PRTArtificial Sequencebinding polypeptide
452Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His
1 5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln
Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr
Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr Glu Gly Pro Asn Gly His Glu
Leu Ile Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
8545386PRTArtificial Sequencebinding polypeptide 453Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Met Ala Gln Asn Val Arg Glu Leu Leu Thr Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
8545486PRTArtificial Sequencebinding polypeptide 454Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Val Thr Gln Asn Gly His Glu Leu Ile Thr Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 8545586PRTArtificial
Sequencebinding polypeptide 455Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Val Thr Gln Asn Asp His Glu Leu Phe Thr Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 8545686PRTArtificial Sequencebinding polypeptide
456Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His
1 5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln
Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr
Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr Val Ala Gln Asn Gly His Glu
Leu Leu Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
8545786PRTArtificial Sequencebinding polypeptide 457Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Val Ala Gln Asn Asp Arg Gln Leu Phe Thr Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 8545886PRTArtificial
Sequencebinding polypeptide 458Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Val Gly Pro Asn Asp Arg Glu Leu Ile Val Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 8545986PRTArtificial Sequencebinding polypeptide
459Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His
1 5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln
Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr
Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr Val Ala Gln Asn Glu His Glu
Leu Leu Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
8546086PRTArtificial Sequencebinding polypeptide 460Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Leu Ala Gln Asn Asn His Glu Leu Ile Thr Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 8546186PRTArtificial
Sequencebinding polypeptide 461Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Glu Ala His His Gly His Glu Leu Met Ile Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 8546286PRTArtificial Sequencebinding polypeptide
462Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His
1 5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln
Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr
Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr Gly Asp His Asn Asp Arg Glu
Leu Met Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
8546394PRTArtificial Sequencebinding polypeptide 463Val Ser Asp Val
Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr 1 5 10 15Ser Leu
Leu Ile Ser Trp Arg His Pro His Phe Pro Thr Arg Tyr Tyr 20 25 30Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 35 40
45Thr Val Pro Leu Gln Pro Pro Thr Ala Thr Ile Ser Gly Leu Lys Pro
50 55 60Gly Val Asp Tyr Thr Ile Thr Gly Tyr Ala Val Thr Gly Gly Gln
Met65 70 75 80Asn Arg Val Leu Met Thr Pro Ile Ser Ile Asn Tyr Arg
Thr 85 9046486PRTArtificial Sequencebinding polypeptide 464Glu Val
Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10
15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly
20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro
Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile
Thr Gly 50 55 60Tyr Ala Val Thr Leu Ala His Asn Asp Arg Glu Leu Ile
Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 8546586PRTArtificial
Sequencebinding polypeptide 465Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Val Pro Gln Asn Gly His Glu Leu Ile Thr Met Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 8546686PRTArtificial Sequencebinding polypeptide
466Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His
1 5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln
Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr
Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr Leu Ala Gln Asn Asp His Glu
Leu Phe Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
8546786PRTArtificial Sequencebinding polypeptide 467Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Val Asp Gln Asn Asp His Glu Leu Phe Thr Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 8546894PRTArtificial
Sequencebinding polypeptide 468Val Ser Asp Val Pro Arg Asp Leu Glu
Val Val Ala Ala Thr Pro Thr 1 5 10 15Ser Leu Leu Ile Ser Trp Arg
His Pro His Phe Pro Thr Arg Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 35 40 45Thr Val Pro Leu Gln
Pro Pro Thr Ala Thr Ile Ser Gly Leu Lys Pro 50 55 60Gly Val Asp Tyr
Thr Ile Thr Gly Tyr Ala Val Thr Val Ala Trp Asn65 70 75 80Asp His
Met Leu Met Thr Pro Ile Ser Ile Asn Tyr Arg Thr 85
9046994PRTArtificial Sequencebinding polypeptide 469Val Ser Asp Val
Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr 1 5 10 15Ser Leu
Leu Ile Ser Trp Arg His Pro His Phe Pro Thr Arg Tyr Tyr 20 25 30Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 35 40
45Thr Val Pro Leu Gln Pro Pro Thr Ala Thr Ile Ser Gly Leu Lys Pro
50 55 60Gly Val Asp Tyr Thr Ile Thr Gly Tyr Ala Val Thr Ser Gly His
Asn65 70 75 80Asp His Met Leu Met Ile Pro Ile Ser Ile Asn Tyr Arg
Thr 85 9047086PRTArtificial Sequencebinding polypeptide 470Glu Val
Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10
15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly
20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro
Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile
Thr Gly 50 55 60Tyr Ala Val Thr Leu Ala Gln Asn Gly His Val Leu Ile
Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 8547186PRTArtificial
Sequencebinding polypeptide 471Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Val Thr His Asn Asp His Glu Leu Leu Thr Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 8547286PRTArtificial Sequencebinding polypeptide
472Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His
1 5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln
Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr
Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr Val Gly Gln Asn Asp His Glu
Leu Met Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
8547386PRTArtificial Sequencebinding polypeptide 473Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Leu Ala Gln Asn Asp His Glu Ile Met Thr Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 8547486PRTArtificial
Sequencebinding polypeptide 474Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Leu Ala Gln Asn Asp His Glu Leu Ile Thr Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 8547586PRTArtificial Sequencebinding polypeptide
475Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His
1 5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln
Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr
Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr Val Ser Gln Gln Asn His Glu
Leu Leu Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
8547686PRTArtificial Sequencebinding polypeptide 476Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Val Ala Gln Asn Asp His Glu Leu Met Thr Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 8547786PRTArtificial
Sequencebinding polypeptide 477Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Val Ala Tyr Asn Glu His Glu Leu Tyr Thr Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 8547886PRTArtificial Sequencebinding polypeptide
478Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His
1 5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln
Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr
Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr Val Ala Gln His Asp His Glu
Leu Leu Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
8547986PRTArtificial Sequencebinding polypeptide 479Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Val Gly Gln Asn Asp Gln Glu Leu Leu Thr Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 8548086PRTArtificial
Sequencebinding polypeptide 480Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile
Thr Gly 50 55 60Tyr Ala Val Thr Val Ala Arg Asn Asp His Glu Leu Met
Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 8548186PRTArtificial
Sequencebinding polypeptide 481Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Val Gly Pro Thr Asp His Glu Leu Leu Thr Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 8548286PRTArtificial Sequencebinding polypeptide
482Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His
1 5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln
Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr
Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr Val Gly Leu Thr Asp His Val
Leu Leu Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
8548386PRTArtificial Sequencebinding polypeptide 483Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Val Ala Gln Asp Asp His Glu Leu Phe Thr Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 8548486PRTArtificial
Sequencebinding polypeptide 484Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Leu Ala Gln Asn Asp His Glu Leu Phe Thr Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 8548586PRTArtificial Sequencebinding polypeptide
485Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His
1 5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln
Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr
Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr Val Gly Trp Asn Asp His Glu
Leu Ile Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
8548686PRTArtificial Sequencebinding polypeptide 486Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Val Ala Gln Asn Asp His Glu Leu Phe Thr Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 8548786PRTArtificial
Sequencebinding polypeptide 487Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Leu Gly Gln Glu Asn Gln Glu Leu Ile Thr Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 8548894PRTArtificial Sequencebinding polypeptide
488Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr
1 5 10 15Ser Leu Leu Ile Ser Trp Arg His Pro His Phe Pro Thr Arg
Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val
Gln Glu Phe 35 40 45Thr Val Pro Leu Gln Pro Pro Thr Ala Thr Ile Ser
Gly Leu Lys Pro 50 55 60Gly Val Asp Tyr Thr Ile Thr Gly Tyr Ala Val
Thr Leu Ala Pro Ser65 70 75 80Ala Arg Glu Leu Met Thr Pro Ile Ser
Ile Asn Tyr Arg Thr 85 9048986PRTArtificial Sequencebinding
polypeptide 489Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser
Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr
Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val
Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly
Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr Val Val His Asn
Gly His Glu Ile Leu Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
8549086PRTArtificial Sequencebinding polypeptide 490Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Met Gly Tyr Glu Asp His Glu Leu Ile Thr Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 8549194PRTArtificial
Sequencebinding polypeptide 491Val Ser Asp Val Pro Arg Asp Leu Glu
Val Val Ala Ala Thr Pro Thr 1 5 10 15Ser Leu Leu Ile Ser Trp Arg
His Pro His Phe Pro Thr Arg Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 35 40 45Thr Val Pro Leu Gln
Pro Pro Thr Ala Thr Ile Ser Gly Leu Lys Pro 50 55 60Gly Val Asp Tyr
Thr Ile Thr Gly Tyr Ala Val Thr Glu Gly Tyr Gln65 70 75 80Asn His
Glu Leu Ser Val Pro Ile Ser Ile Asn Tyr Arg Thr 85
9049286PRTArtificial Sequencebinding polypeptide 492Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Val Asp Gln Asn Asp His Glu Leu Phe Thr Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 8549386PRTArtificial
Sequencebinding polypeptide 493Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Val Ala Gln Ser Asp His Glu Leu Met Thr Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 8549486PRTArtificial Sequencebinding polypeptide
494Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His
1 5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln
Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr
Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr Val Gly Gln Asn Asp His Glu
Leu Ile Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
8549586PRTArtificial Sequencebinding polypeptide 495Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Val Ala Gln Asn Asp His Glu Leu Met Thr Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 8549686PRTArtificial
Sequencebinding polypeptide 496Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Val Ala Gln Asn Gly His Glu Leu Ile Thr Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 8549786PRTArtificial Sequencebinding polypeptide
497Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His
1 5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln
Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr
Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr Arg Ala Gln Asn Asp His Glu
Leu Ile Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
8549886PRTArtificial Sequencebinding polypeptide 498Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Val Ala Gln Ser Asn His Glu Leu Met Thr Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 8549986PRTArtificial
Sequencebinding polypeptide 499Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Val Ala Gln Asn Asp Arg Glu Leu Ile Thr Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 8550086PRTArtificial Sequencebinding polypeptide
500Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His
1 5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln
Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr
Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr Leu Thr His Asn Glu Gln Tyr
Leu Phe Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
8550194PRTArtificial Sequencebinding polypeptide 501Val Ser Asp Val
Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr 1 5 10 15Ser Leu
Leu Ile Ser Trp Arg His Pro His Phe Pro Thr Arg Tyr Tyr 20 25 30Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 35 40
45Thr Val Pro Leu Gln Pro Pro Thr Ala Thr Ile Ser Gly Leu Lys Pro
50 55 60Gly Val Asp Tyr Thr Ile Thr Gly Tyr Ala Val Thr Glu Ile Tyr
Asn65 70 75 80Asp His Glu Leu Met Thr Pro Ile Ser Ile Asn Tyr Arg
Thr 85 9050286PRTArtificial Sequencebinding polypeptide 502Glu Val
Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10
15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly
20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro
Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile
Thr Gly 50 55 60Tyr Ala Val Thr Met Ala Gln Asn Asp His Glu Leu Ile
Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 8550386PRTArtificial
Sequencebinding polypeptide 503Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Val Ser Gln Tyr Gly His Glu Leu Ile Thr Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 8550486PRTArtificial Sequencebinding polypeptide
504Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His
1 5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln
Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr
Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr Val Ala Lys Asn Asp His Glu
Leu Ile Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
8550586PRTArtificial Sequencebinding polypeptide 505Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Val Ala Gln Asn Asn His Glu Leu Ile Thr Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 8550686PRTArtificial
Sequencebinding polypeptide 506Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Val Ala Gln His Asp His Glu Leu Leu Thr Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 8550794PRTArtificial Sequencebinding polypeptide
507Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr
1 5 10 15Ser Leu Leu Ile Ser Trp Arg His Pro His Phe Pro Thr Arg
Tyr Tyr
20 25 30Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu
Phe 35 40 45Thr Val Pro Leu Gln Pro Pro Thr Ala Thr Ile Ser Gly Leu
Lys Pro 50 55 60Gly Val Asp Tyr Thr Ile Thr Gly Tyr Ala Val Thr Leu
Ser His Tyr65 70 75 80Gly Lys Glu Leu Arg Thr Pro Ile Ser Ile Asn
Tyr Arg Thr 85 9050886PRTArtificial Sequencebinding polypeptide
508Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His
1 5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln
Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr
Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr Val Ala Gln Asn Ala His Glu
Leu Met Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
8550986PRTArtificial Sequencebinding polypeptide 509Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Leu Gly Gln Asn Asp His Glu Leu Leu Thr Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 8551086PRTArtificial
Sequencebinding polypeptide 510Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Val Ala Gln Asn Asp His Glu Leu Lys Thr Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 8551186PRTArtificial Sequencebinding polypeptide
511Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His
1 5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln
Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr
Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr Gly Glu Gln Asn Asp Tyr Glu
Leu Leu Val Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
8551286PRTArtificial Sequencebinding polypeptide 512Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His
Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40
45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly
50 55 60Tyr Ala Val Thr Leu Thr Gln His Asp His Glu Leu Leu Thr Pro
Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 8551386PRTArtificial
Sequencebinding polypeptide 513Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Met Ala Gln Asn Asp His Glu Leu Ile Thr Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 8551494PRTArtificial Sequencebinding polypeptide
514Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr
1 5 10 15Ser Leu Leu Ile Ser Trp Arg His Pro His Phe Pro Thr Arg
Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val
Gln Glu Phe 35 40 45Thr Val Pro Leu Gln Pro Pro Thr Ala Thr Ile Ser
Gly Leu Lys Pro 50 55 60Gly Val Asp Tyr Thr Ile Thr Gly Tyr Ala Val
Thr Glu Ala Pro Asn65 70 75 80Gly Arg Glu Leu Arg Thr Pro Ile Ser
Ile Asn Tyr Arg Thr 85 9051594PRTArtificial Sequencebinding
polypeptide 515Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala
Thr Pro Thr 1 5 10 15Ser Leu Leu Ile Ser Trp Arg His Pro His Phe
Pro Thr Arg Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn
Ser Pro Val Gln Glu Phe 35 40 45Thr Val Pro Leu Gln Pro Pro Thr Ala
Thr Ile Ser Gly Leu Lys Pro 50 55 60Gly Val Asp Tyr Thr Ile Thr Gly
Tyr Ala Val Thr Val Gly Pro Thr65 70 75 80Asp His Glu Leu Leu Thr
Pro Ile Ser Ile Asn Tyr Arg Thr 85 9051686PRTArtificial
Sequencebinding polypeptide 516Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Arg His 1 5 10 15Pro His Phe Pro Thr Arg Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Leu Gln Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly
Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr
Val Gly Gln Tyr Asp His Glu Leu Ile Thr Pro Ile65 70 75 80Ser Ile
Asn Tyr Arg Thr 8551786PRTArtificial Sequencebinding polypeptide
517Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His
1 5 10 15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln
Pro Pro Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr
Thr Ile Thr Gly 50 55 60Tyr Ala Val Thr Val Ala Gln Asp Glu His Glu
Leu Ile Thr Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr
8551894PRTArtificial Sequencebinding polypeptide 518Val Ser Asp Val
Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr 1 5 10 15Ser Leu
Leu Ile Ser Trp Arg His Pro His Phe Pro Thr Arg Tyr Tyr 20 25 30Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 35 40
45Thr Val Pro Leu Gln Pro Pro Thr Ala Thr Ile Ser Gly Leu Lys Pro
50 55 60Gly Val Asp Tyr Thr Ile Thr Gly Tyr Ala Val Thr Asp Ala Gln
Asn65 70 75 80Val Gln Ala Pro Ile Ala Gln Ile Ser Ile Asn Tyr Arg
Thr 85 9051994PRTArtificial Sequencebinding polypeptide 519Val Ser
Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr 1 5 10
15Ser Leu Leu Ile Ser Trp Arg His Pro His Phe Pro Thr Arg Tyr Tyr
20 25 30Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu
Phe 35 40 45Thr Val Pro Leu Gln Pro Pro Thr Ala Thr Ile Ser Gly Leu
Lys Pro 50 55 60Gly Val Asp Tyr Thr Ile Thr Gly Tyr Ala Val Thr Ser
Gly Gln Asn65 70 75 80Asp His Ala Leu Leu Ile Pro Ile Ser Ile Asn
Tyr Arg Thr 85 9052094PRTArtificial Sequencebinding polypeptide
520Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr
1 5 10 15Ser Leu Leu Ile Ser Trp Arg His Pro His Phe Pro Thr Arg
Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val
Gln Glu Phe 35 40 45Thr Val Pro Leu Gln Pro Pro Thr Ala Thr Ile Ser
Gly Leu Lys Pro 50 55 60Gly Val Asp Tyr Thr Ile Thr Gly Tyr Ala Val
Thr Glu Asp Ile Arg65 70 75 80Val Leu Trp Leu Asn Thr Thr Ile Ser
Ile Asn Tyr Arg Thr 85 9052194PRTArtificial Sequencebinding
polypeptide 521Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala
Thr Pro Thr 1 5 10 15Ser Leu Leu Ile Ser Trp Arg His Pro His Phe
Pro Thr Arg Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn
Ser Pro Val Gln Glu Phe 35 40 45Thr Val Pro Leu Gln Pro Pro Thr Ala
Thr Ile Ser Gly Leu Lys Pro 50 55 60Gly Val Asp Tyr Thr Ile Thr Gly
Tyr Ala Val Thr Trp Asp Gln Asn65 70 75 80Gly His Val Leu Leu Thr
Pro Ile Ser Ile Asn Tyr Arg Thr 85 9052294PRTArtificial
Sequencebinding polypeptide 522Val Ser Asp Val Pro Arg Asp Leu Glu
Val Val Ala Ala Thr Pro Thr 1 5 10 15Ser Leu Leu Ile Ser Trp Arg
His Pro His Phe Pro Thr Arg Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 35 40 45Thr Val Pro Leu Gln
Pro Pro Thr Ala Thr Ile Ser Gly Leu Lys Pro 50 55 60Gly Val Asp Tyr
Thr Ile Thr Gly Tyr Ala Val Thr Leu Gly Leu Arg65 70 75 80Asp Arg
Glu Leu Phe Val Pro Ile Ser Ile Asn Tyr Arg Thr 85
9052394PRTArtificial Sequencebinding polypeptide 523Val Ser Asp Val
Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr 1 5 10 15Ser Leu
Leu Ile Ser Trp Arg His Pro His Phe Pro Thr Arg Tyr Tyr 20 25 30Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 35 40
45Thr Val Pro Leu Gln Pro Pro Thr Ala Thr Ile Ser Gly Leu Lys Pro
50 55 60Gly Val Asp Tyr Thr Ile Thr Gly Tyr Ala Val Thr Val Glu Pro
Asn65 70 75 80Gly His Lys Leu Ala Ile Pro Ile Ser Ile Asn Tyr Arg
Thr 85 9052486PRTArtificial Sequencebinding polypeptide 524Glu Val
Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Arg His 1 5 10
15Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly
20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Leu Gln Pro Pro
Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile
Thr Gly 50 55 60Tyr Ala Val Thr Phe Gly Gln Asn Gly Lys Glu Phe Arg
Ile Pro Ile65 70 75 80Ser Ile Asn Tyr Arg Thr 8552594PRTArtificial
Sequencebinding polypeptide 525Val Ser Asp Val Pro Arg Asp Leu Glu
Val Val Ala Ala Thr Pro Thr 1 5 10 15Ser Leu Leu Ile Ser Trp Arg
His Pro His Phe Pro Thr Arg Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 35 40 45Thr Val Pro Leu Gln
Pro Pro Thr Ala Thr Ile Ser Gly Leu Lys Pro 50 55 60Gly Val Asp Tyr
Thr Ile Thr Gly Tyr Ala Val Thr Ser Gly His Asn65 70 75 80Gly His
Glu Val Met Thr Pro Ile Ser Ile Asn Tyr Arg Thr 85
9052694PRTArtificial Sequencebinding polypeptide 526Val Ser Asp Val
Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr 1 5 10 15Ser Leu
Leu Ile Ser Trp Arg His Pro His Phe Pro Thr Arg Tyr Tyr 20 25 30Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 35 40
45Thr Val Pro Leu Gln Pro Pro Thr Ala Thr Ile Ser Gly Leu Lys Pro
50 55 60Gly Val Asp Tyr Thr Ile Thr Gly Tyr Ala Val Thr Leu Gly Trp
Asn65 70 75 80Asp His Glu Leu Tyr Ile Pro Ile Ser Ile Asn Tyr Arg
Thr 85 9052794PRTArtificial Sequencebinding polypeptide 527Val Ser
Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr 1 5 10
15Ser Leu Leu Ile Ser Trp Arg His Pro His Phe Pro Thr Arg Tyr Tyr
20 25 30Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu
Phe 35 40 45Thr Val Pro Leu Gln Pro Pro Thr Ala Thr Ile Ser Gly Leu
Lys Pro 50 55 60Gly Val Asp Tyr Thr Ile Thr Gly Tyr Ala Val Thr Leu
Gly Lys Asp65 70 75 80Val Arg Glu Leu Leu Thr Pro Ile Ser Ile Asn
Tyr Arg Thr 85 9052894PRTArtificial Sequencebinding polypeptide
528Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr
1 5 10 15Ser Leu Leu Ile Ser Trp Arg His Pro His Phe Pro Thr Arg
Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val
Gln Glu Phe 35 40 45Thr Val Pro Leu Gln Pro Pro Thr Ala Thr Ile Ser
Gly Leu Lys Pro 50 55 60Gly Val Asp Tyr Thr Ile Thr Gly Tyr Ala Val
Thr Leu Ala Leu Phe65 70 75 80Asp His Glu Leu Leu Thr Pro Ile Ser
Ile Asn Tyr Arg Thr 85 90
* * * * *
References